rabbit anti goat igg hrp conjugated antibody  (Thermo Fisher)


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    Structured Review

    Thermo Fisher rabbit anti goat igg hrp conjugated antibody
    The immunized mouse serum <t>IgG</t> (H+L) titer and IgG subtypes were detected using the indirect ELISA assay. (A) IgG titers of BALB/c mice immunized with rEF-Tu, rHSP70, Mo extracts and PBS sera of each groups were collected on days 3, 7, 14, 21, 28, 35, 42, 49, 56, 63 and 70 DAI. (B) Determination of IgG subtypes in sera of the immunized mice. The sera of each group were collected at 35 DAI. ELISA plates were coated with purified rEF-Tu proteins or rHSP70 proteins or Mo extracts of M . ovipneumoniae wild strain Mo-1 at a concentration of 100 ng per well. Anti-mouse IgG (H+L) or IgG1 or IgG2a <t>HRP-conjugated</t> antibodies were used as secondary antibodies. Asterisks indicates the results of the One-Way ANOVA using the Tukey test, compared with the PBS negative control group, with P
    Rabbit Anti Goat Igg Hrp Conjugated Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 2024 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti goat igg hrp conjugated antibody/product/Thermo Fisher
    Average 85 stars, based on 2024 article reviews
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    rabbit anti goat igg hrp conjugated antibody - by Bioz Stars, 2020-02
    85/100 stars

    Images

    1) Product Images from "Elongation Factor Tu and Heat Shock Protein 70 Are Membrane-Associated Proteins from Mycoplasma ovipneumoniae Capable of Inducing Strong Immune Response in Mice"

    Article Title: Elongation Factor Tu and Heat Shock Protein 70 Are Membrane-Associated Proteins from Mycoplasma ovipneumoniae Capable of Inducing Strong Immune Response in Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0161170

    The immunized mouse serum IgG (H+L) titer and IgG subtypes were detected using the indirect ELISA assay. (A) IgG titers of BALB/c mice immunized with rEF-Tu, rHSP70, Mo extracts and PBS sera of each groups were collected on days 3, 7, 14, 21, 28, 35, 42, 49, 56, 63 and 70 DAI. (B) Determination of IgG subtypes in sera of the immunized mice. The sera of each group were collected at 35 DAI. ELISA plates were coated with purified rEF-Tu proteins or rHSP70 proteins or Mo extracts of M . ovipneumoniae wild strain Mo-1 at a concentration of 100 ng per well. Anti-mouse IgG (H+L) or IgG1 or IgG2a HRP-conjugated antibodies were used as secondary antibodies. Asterisks indicates the results of the One-Way ANOVA using the Tukey test, compared with the PBS negative control group, with P
    Figure Legend Snippet: The immunized mouse serum IgG (H+L) titer and IgG subtypes were detected using the indirect ELISA assay. (A) IgG titers of BALB/c mice immunized with rEF-Tu, rHSP70, Mo extracts and PBS sera of each groups were collected on days 3, 7, 14, 21, 28, 35, 42, 49, 56, 63 and 70 DAI. (B) Determination of IgG subtypes in sera of the immunized mice. The sera of each group were collected at 35 DAI. ELISA plates were coated with purified rEF-Tu proteins or rHSP70 proteins or Mo extracts of M . ovipneumoniae wild strain Mo-1 at a concentration of 100 ng per well. Anti-mouse IgG (H+L) or IgG1 or IgG2a HRP-conjugated antibodies were used as secondary antibodies. Asterisks indicates the results of the One-Way ANOVA using the Tukey test, compared with the PBS negative control group, with P

    Techniques Used: Indirect ELISA, Mouse Assay, Enzyme-linked Immunosorbent Assay, Purification, Concentration Assay, Negative Control

    2) Product Images from "Expression and regulation of avian beta-defensin 8 protein in immune tissues and cell lines of chickens"

    Article Title: Expression and regulation of avian beta-defensin 8 protein in immune tissues and cell lines of chickens

    Journal: Asian-Australasian Journal of Animal Sciences

    doi: 10.5713/ajas.17.0836

    Determination of AvBD8 protein expression in immune cell lines by performing immunocytochemical analysis. Cultured chicken CU91 T-, DT40 B-, HD11 macrophage-, and OU2 fibroblast-cell lines were incubated with the rabbit anti-AvBD8 primary antibody, followed by incubation with the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody, and were counterstained with 4′,6-diamidino-2-phenylindole. Control cells were incubated with secondary antibody only. AvBD8, avian beta-defensin 8; IgG, immunoglobulin G. Scale bar: 20 μm.
    Figure Legend Snippet: Determination of AvBD8 protein expression in immune cell lines by performing immunocytochemical analysis. Cultured chicken CU91 T-, DT40 B-, HD11 macrophage-, and OU2 fibroblast-cell lines were incubated with the rabbit anti-AvBD8 primary antibody, followed by incubation with the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody, and were counterstained with 4′,6-diamidino-2-phenylindole. Control cells were incubated with secondary antibody only. AvBD8, avian beta-defensin 8; IgG, immunoglobulin G. Scale bar: 20 μm.

    Techniques Used: Expressing, Cell Culture, Incubation

    Determination of AvBD8 protein expression in immune tissues of male WL chickens by performing immunohistochemical analysis. Immunohistochemical analysis was performed to assess AvBD8 protein expression in the thymus, spleen, liver, small intestine, and ceca of male WL chickens aged 25 weeks. Frozen sections were incubated with the rabbit anti-AvBD8 primary antibody, followed by incubation with the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody, and were counterstained with 4′,6-diamidino-2-phenylindole. Control sections were incubated with secondary antibody only. Boxed region in the middle column is enlarged in the right column. AvBD8, avian beta-defensin 8; WL, White Leghorn; IgG, immunoglobulin G. Scale bar: 200 μm (left and middle columns) and 50 μm (right column). Arrowheads indicate strong AvBD8 signal in the intestinal mucosal layer.
    Figure Legend Snippet: Determination of AvBD8 protein expression in immune tissues of male WL chickens by performing immunohistochemical analysis. Immunohistochemical analysis was performed to assess AvBD8 protein expression in the thymus, spleen, liver, small intestine, and ceca of male WL chickens aged 25 weeks. Frozen sections were incubated with the rabbit anti-AvBD8 primary antibody, followed by incubation with the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody, and were counterstained with 4′,6-diamidino-2-phenylindole. Control sections were incubated with secondary antibody only. Boxed region in the middle column is enlarged in the right column. AvBD8, avian beta-defensin 8; WL, White Leghorn; IgG, immunoglobulin G. Scale bar: 200 μm (left and middle columns) and 50 μm (right column). Arrowheads indicate strong AvBD8 signal in the intestinal mucosal layer.

    Techniques Used: Expressing, Immunohistochemistry, Incubation

    3) Product Images from "Evaluation of a Plasmodium-Specific Carrier Protein To Enhance Production of Recombinant Pfs25, a Leading Transmission-Blocking Vaccine Candidate"

    Article Title: Evaluation of a Plasmodium-Specific Carrier Protein To Enhance Production of Recombinant Pfs25, a Leading Transmission-Blocking Vaccine Candidate

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00486-17

    Pf MSP8 is expressed during P. falciparum gametocyte development but is absent on the surface of activated macrogametes. (A) Detection of Pf MSP8 expression in fixed and permeabilized P. falciparum gametocytes (stages II to V) by an immunofluorescence assay with rabbit anti-r Pf MSP8 IgG or control IgG followed by FITC-conjugated secondary antibodies. DAPI was used to stain parasite DNA. (B) Analysis of Pf MSP8 expression on activated, live P. falciparum macrogametes by an immunofluorescence assay, as described above, with rabbit anti-r Pf MSP8 IgG. Samples were costained with MAb 4B7, which is specific for Pf s25 (MAb 4B7), followed by TRITC-conjugated secondary IgG. DIC, differential interference contrast.
    Figure Legend Snippet: Pf MSP8 is expressed during P. falciparum gametocyte development but is absent on the surface of activated macrogametes. (A) Detection of Pf MSP8 expression in fixed and permeabilized P. falciparum gametocytes (stages II to V) by an immunofluorescence assay with rabbit anti-r Pf MSP8 IgG or control IgG followed by FITC-conjugated secondary antibodies. DAPI was used to stain parasite DNA. (B) Analysis of Pf MSP8 expression on activated, live P. falciparum macrogametes by an immunofluorescence assay, as described above, with rabbit anti-r Pf MSP8 IgG. Samples were costained with MAb 4B7, which is specific for Pf s25 (MAb 4B7), followed by TRITC-conjugated secondary IgG. DIC, differential interference contrast.

    Techniques Used: Expressing, Immunofluorescence, Staining

    4) Product Images from "Tetraspanin CD82 interaction with cholesterol promotes extracellular vesicle–mediated release of ezrin to inhibit tumour cell movement"

    Article Title: Tetraspanin CD82 interaction with cholesterol promotes extracellular vesicle–mediated release of ezrin to inhibit tumour cell movement

    Journal: Journal of Extracellular Vesicles

    doi: 10.1080/20013078.2019.1692417

    CD82 and its cholesterol-binding differentially regulate cellular release of EVs. (a) Extracellular staining by filipin and Alexa488-conjugated CTxb in Du145 transfectants. Equal number of the cells were cultured on glass coverslips for 2 days, then fixed and labelled with filipin or Alexa488-conjugated CTxb. For filipin staining, intercellular regions were imaged. For CTxb staining, pericellular regions were imaged. Scale bar: 10 µm. (b) Distributions of Annexin V and Annexin A2 in Du145 transfectant cells. Alexa488-conjugated recombinant Annexin V was used for phosphatidylserine labelling, while Annexin-A2 Ab was used for Annexin-A2 staining. Scale bar: 10 μm. (c) Colocalization of Ezrin with GM1 or Annexin A2 in EVs. For Ezrin and GM1 co-staining, the cells were labelled with the Abs, Alexa488-conjugated CTxB and DAPI. For Ezrin and Annexin A2 co-staining, the cells were incubated sequentially with the primary Abs, Cy3-conjugated donkey anti-goat IgG, normal goat IgG and Alexa594-conjugated goat anti-mouse IgG. Images were obtained by confocal microscopy. Scale bar: 10 µm. (d) The cells were seeded in six-well plate at 50% confluence and cultured in DMEM containing 1% exosome-depleted FBS for 2 – 3 days. The culture supernatants were collected, spun at 2000 × g for 10 min to remove cell debris, and then analysed with NanoSight instrument for EV number and size. Data are presented as mean ± SD (n = 3 individual experiments). *: p
    Figure Legend Snippet: CD82 and its cholesterol-binding differentially regulate cellular release of EVs. (a) Extracellular staining by filipin and Alexa488-conjugated CTxb in Du145 transfectants. Equal number of the cells were cultured on glass coverslips for 2 days, then fixed and labelled with filipin or Alexa488-conjugated CTxb. For filipin staining, intercellular regions were imaged. For CTxb staining, pericellular regions were imaged. Scale bar: 10 µm. (b) Distributions of Annexin V and Annexin A2 in Du145 transfectant cells. Alexa488-conjugated recombinant Annexin V was used for phosphatidylserine labelling, while Annexin-A2 Ab was used for Annexin-A2 staining. Scale bar: 10 μm. (c) Colocalization of Ezrin with GM1 or Annexin A2 in EVs. For Ezrin and GM1 co-staining, the cells were labelled with the Abs, Alexa488-conjugated CTxB and DAPI. For Ezrin and Annexin A2 co-staining, the cells were incubated sequentially with the primary Abs, Cy3-conjugated donkey anti-goat IgG, normal goat IgG and Alexa594-conjugated goat anti-mouse IgG. Images were obtained by confocal microscopy. Scale bar: 10 µm. (d) The cells were seeded in six-well plate at 50% confluence and cultured in DMEM containing 1% exosome-depleted FBS for 2 – 3 days. The culture supernatants were collected, spun at 2000 × g for 10 min to remove cell debris, and then analysed with NanoSight instrument for EV number and size. Data are presented as mean ± SD (n = 3 individual experiments). *: p

    Techniques Used: Binding Assay, Staining, Cell Culture, Transfection, Recombinant, Incubation, Confocal Microscopy

    5) Product Images from "US3 Kinase-Mediated Phosphorylation of Tegument Protein VP8 Plays a Critical Role in the Cellular Localization of VP8 and Its Effect on the Lipid Metabolism of Bovine Herpesvirus 1-Infected Cells"

    Article Title: US3 Kinase-Mediated Phosphorylation of Tegument Protein VP8 Plays a Critical Role in the Cellular Localization of VP8 and Its Effect on the Lipid Metabolism of Bovine Herpesvirus 1-Infected Cells

    Journal: Journal of Virology

    doi: 10.1128/JVI.02151-18

    Cytoplasmic localization of VP8 at a late stage of BoHV-1 infection requires US3. (A to C) MDBK cells were infected with the indicated viruses at an MOI of 5 or mock-infected. Cells were processed for immunofluorescence at 4 and 8 hpi. VP8 was detected with monoclonal anti-VP8 antibody followed by Alexa Fluor 488-conjugated goat anti-mouse IgG. US3 was identified with polyclonal anti-US3 antibody followed by Alexa Fluor 633-conjugated goat anti-rabbit IgG. The cell nuclei were identified with DAPI. (D) Virus-infected MDBK cell lysates were analyzed by Western blotting. VP8 and US3 were detected with mouse anti-VP8 and rabbit anti-US3 antibodies followed by IRDye 800CW goat anti-mouse IgG and IRDye 680RD goat anti-rabbit IgG, respectively.
    Figure Legend Snippet: Cytoplasmic localization of VP8 at a late stage of BoHV-1 infection requires US3. (A to C) MDBK cells were infected with the indicated viruses at an MOI of 5 or mock-infected. Cells were processed for immunofluorescence at 4 and 8 hpi. VP8 was detected with monoclonal anti-VP8 antibody followed by Alexa Fluor 488-conjugated goat anti-mouse IgG. US3 was identified with polyclonal anti-US3 antibody followed by Alexa Fluor 633-conjugated goat anti-rabbit IgG. The cell nuclei were identified with DAPI. (D) Virus-infected MDBK cell lysates were analyzed by Western blotting. VP8 and US3 were detected with mouse anti-VP8 and rabbit anti-US3 antibodies followed by IRDye 800CW goat anti-mouse IgG and IRDye 680RD goat anti-rabbit IgG, respectively.

    Techniques Used: Infection, Immunofluorescence, Western Blot

    The nuclear export of VP8 is sensitive to LMB. MDBK cells were infected with BoHV-1. Culture medium supplemented or not with LMB (20 nM) was applied to cells at 1 hpi. Medium with or without LMB was renewed every 2 h. Cells were fixed at 4, 6, and 8 hpi and incubated with monoclonal anti-VP8 antibody followed by Alexa Fluor 488-conjugated goat anti-mouse IgG. The cell nuclei were identified with DAPI.
    Figure Legend Snippet: The nuclear export of VP8 is sensitive to LMB. MDBK cells were infected with BoHV-1. Culture medium supplemented or not with LMB (20 nM) was applied to cells at 1 hpi. Medium with or without LMB was renewed every 2 h. Cells were fixed at 4, 6, and 8 hpi and incubated with monoclonal anti-VP8 antibody followed by Alexa Fluor 488-conjugated goat anti-mouse IgG. The cell nuclei were identified with DAPI.

    Techniques Used: Infection, Incubation

    Phosphorylation by CK2 does not change the nuclear localization of VP8. (A) Mutating CK2-phosphorylated residues in VP8 does not affect the nuclear localization of VP8. EBTr cells were transfected with pFLAG-VP8 or pFLAG-VP8-M65-107 and fixed for immunofluorescent staining. (B) Overexpression of CK2α does not alter the VP8 nuclear localization. Plasmid pFLAG-VP8 or pFLAG-VP8-M65-107 was cotransfected with pCK2α-HA or individually transfected into COS-7 cells. The cells were fixed for immunofluorescent staining. VP8 was identified with polyclonal anti-VP8 antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG. CK2α-HA was identified with monoclonal anti-HA antibody followed by Alexa Fluor 633-conjugated goat anti-mouse IgG. The cell nuclei were identified with DAPI.
    Figure Legend Snippet: Phosphorylation by CK2 does not change the nuclear localization of VP8. (A) Mutating CK2-phosphorylated residues in VP8 does not affect the nuclear localization of VP8. EBTr cells were transfected with pFLAG-VP8 or pFLAG-VP8-M65-107 and fixed for immunofluorescent staining. (B) Overexpression of CK2α does not alter the VP8 nuclear localization. Plasmid pFLAG-VP8 or pFLAG-VP8-M65-107 was cotransfected with pCK2α-HA or individually transfected into COS-7 cells. The cells were fixed for immunofluorescent staining. VP8 was identified with polyclonal anti-VP8 antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG. CK2α-HA was identified with monoclonal anti-HA antibody followed by Alexa Fluor 633-conjugated goat anti-mouse IgG. The cell nuclei were identified with DAPI.

    Techniques Used: Transfection, Staining, Over Expression, Plasmid Preparation

    Nuclear lipid droplets are inversely correlated with phosphorylation of VP8 in infected cells. MDBK cells were infected with Wt BoHV-1, BoHV-1-YmVP8, or ΔUS3-BoHV-1 and fixed for immunostaining. VP8 (WT) and Mut-VP8 were detected with VP8-specific polyclonal antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG. US3 was detected with US3-specific polyclonal antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG. Cells were then incubated with Nile red and DAPI.
    Figure Legend Snippet: Nuclear lipid droplets are inversely correlated with phosphorylation of VP8 in infected cells. MDBK cells were infected with Wt BoHV-1, BoHV-1-YmVP8, or ΔUS3-BoHV-1 and fixed for immunostaining. VP8 (WT) and Mut-VP8 were detected with VP8-specific polyclonal antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG. US3 was detected with US3-specific polyclonal antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG. Cells were then incubated with Nile red and DAPI.

    Techniques Used: Infection, Immunostaining, Incubation

    BFA disperses the cis -Golgi proteins but not the TGN protein. HeLa cells were treated with BFA or DMSO. The BFA was renewed every 2 h. cis -Golgi proteins FTCD and GOLGB1 were detected with mouse anti-FTCD and rabbit anti-GOLGB1 antibodies. TGN protein TGOLN2 was detected with rabbit anti-TGOLN2 antibody. Alexa Fluor 488-conjugated goat anti-mouse IgG and Alexa Fluor 633-conjugated goat anti-rabbit IgG were used as secondary antibodies. The cell nuclei were identified with DAPI.
    Figure Legend Snippet: BFA disperses the cis -Golgi proteins but not the TGN protein. HeLa cells were treated with BFA or DMSO. The BFA was renewed every 2 h. cis -Golgi proteins FTCD and GOLGB1 were detected with mouse anti-FTCD and rabbit anti-GOLGB1 antibodies. TGN protein TGOLN2 was detected with rabbit anti-TGOLN2 antibody. Alexa Fluor 488-conjugated goat anti-mouse IgG and Alexa Fluor 633-conjugated goat anti-rabbit IgG were used as secondary antibodies. The cell nuclei were identified with DAPI.

    Techniques Used:

    US3-mediated phosphorylation promotes the cytoplasmic localization of VP8. (A) EBTr cells were transfected with different (combinations of) plasmids. Cells in panel 1 were transfected with pFLAG-VP8. Cells in panel 2 were cotransfected with pFLAG-VP8 and pUS3-HA. Cells in panel 3 were transfected with pFLAG-VP8-S16A. Cells in panel 4 were cotransfected with pFLAG-VP8-S16A and pUS3-HA. VP8 was identified with monoclonal anti-VP8 antibody followed by Alexa Fluor 488-conjugated goat anti-mouse IgG, and US3 was identified with polyclonal anti-US3 antibody followed by Alexa Fluor 633-conjugated goat anti-rabbit IgG. The cell nuclei were identified with DAPI. (B) Relative quantification of cytoplasmic and nuclear VP8. The software Leica LAS AF Lite was used to analyze confocal pictures. The intensity of VP8 is represented as the fluorescence pixels at a wavelength of 488 nm. The mean PDU are shown in the bar graphs. Error bars represent the SDs. The statistical significance is shown as follows: **, P ≤ 0.01. (C) The nuclear and cytoplasmic fractions were extracted from EBTr cells transfected with the indicated plasmids. VP8 and US3 in the nuclear and cytoplasmic fractions were analyzed by Western blotting. VP8 and US3 were detected with mouse anti-VP8 and rabbit anti-US3 antibodies followed by IRDye 800CW goat anti-mouse IgG and IRDye 680RD goat anti-rabbit IgG, respectively. Nucleolin and tubulin were used to demonstrate the purity of nuclear and cytoplasmic fractions.
    Figure Legend Snippet: US3-mediated phosphorylation promotes the cytoplasmic localization of VP8. (A) EBTr cells were transfected with different (combinations of) plasmids. Cells in panel 1 were transfected with pFLAG-VP8. Cells in panel 2 were cotransfected with pFLAG-VP8 and pUS3-HA. Cells in panel 3 were transfected with pFLAG-VP8-S16A. Cells in panel 4 were cotransfected with pFLAG-VP8-S16A and pUS3-HA. VP8 was identified with monoclonal anti-VP8 antibody followed by Alexa Fluor 488-conjugated goat anti-mouse IgG, and US3 was identified with polyclonal anti-US3 antibody followed by Alexa Fluor 633-conjugated goat anti-rabbit IgG. The cell nuclei were identified with DAPI. (B) Relative quantification of cytoplasmic and nuclear VP8. The software Leica LAS AF Lite was used to analyze confocal pictures. The intensity of VP8 is represented as the fluorescence pixels at a wavelength of 488 nm. The mean PDU are shown in the bar graphs. Error bars represent the SDs. The statistical significance is shown as follows: **, P ≤ 0.01. (C) The nuclear and cytoplasmic fractions were extracted from EBTr cells transfected with the indicated plasmids. VP8 and US3 in the nuclear and cytoplasmic fractions were analyzed by Western blotting. VP8 and US3 were detected with mouse anti-VP8 and rabbit anti-US3 antibodies followed by IRDye 800CW goat anti-mouse IgG and IRDye 680RD goat anti-rabbit IgG, respectively. Nucleolin and tubulin were used to demonstrate the purity of nuclear and cytoplasmic fractions.

    Techniques Used: Transfection, Software, Fluorescence, Western Blot

    The Golgi accumulation of VP8 is sensitive to BFA. (A) MDBK cells infected with BoHV-1 were treated with BFA or DMSO at 1 hpi. Cells were fixed and incubated with monoclonal anti-VP8 antibody followed by Alexa Fluor 488-conjugated goat anti-mouse IgG. The cell nuclei were identified with DAPI. (B) MDBK cells infected with BoHV-1-YVP8 were treated with BFA at 7 hpi. Live cells showing YFP-VP8 were observed from 7 hpi for 20 min.
    Figure Legend Snippet: The Golgi accumulation of VP8 is sensitive to BFA. (A) MDBK cells infected with BoHV-1 were treated with BFA or DMSO at 1 hpi. Cells were fixed and incubated with monoclonal anti-VP8 antibody followed by Alexa Fluor 488-conjugated goat anti-mouse IgG. The cell nuclei were identified with DAPI. (B) MDBK cells infected with BoHV-1-YVP8 were treated with BFA at 7 hpi. Live cells showing YFP-VP8 were observed from 7 hpi for 20 min.

    Techniques Used: Infection, Incubation

    Translocation of VP8 from the nucleus to the cytoplasm in BoHV-1-YVP8-infected cells. EBTr cells transfected with pFLAG-VP8 were cultured for 24 h. The FLAG-VP8-expressing cells were then mock infected or infected with BoHV-1-YVP8 at an MOI of 5. Cells were fixed at the indicated time points for immunofluorescent staining. FLAG-VP8 was detected with monoclonal anti-FLAG antibody followed by Alexa Fluor 633-conjugated goat anti-mouse IgG. YFP-VP8 was tracked through the YFP label. The cell nuclei were identified with DAPI.
    Figure Legend Snippet: Translocation of VP8 from the nucleus to the cytoplasm in BoHV-1-YVP8-infected cells. EBTr cells transfected with pFLAG-VP8 were cultured for 24 h. The FLAG-VP8-expressing cells were then mock infected or infected with BoHV-1-YVP8 at an MOI of 5. Cells were fixed at the indicated time points for immunofluorescent staining. FLAG-VP8 was detected with monoclonal anti-FLAG antibody followed by Alexa Fluor 633-conjugated goat anti-mouse IgG. YFP-VP8 was tracked through the YFP label. The cell nuclei were identified with DAPI.

    Techniques Used: Translocation Assay, Infection, Transfection, Cell Culture, Expressing, Staining

    VP8 is not colocalized with TGN protein AP1G1. (A) GOLGB1 is dispersed by BFA treatment. MDBK cells were treated with BFA. AP1G1 was detected with AP1G1-specific monoclonal antibody followed by Alexa Fluor 488-conjugated goat anti-mouse IgG. GOLGB1 was detected with GOLGB1-specific polyclonal antibody followed by Alexa Fluor 633-conjugated goat anti-rabbit IgG. (B) VP8 is dispersed by BFA treatment. BoHV-1-infected MDBK cells were treated with BFA. VP8 was detected with VP8-specific polyclonal antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG.
    Figure Legend Snippet: VP8 is not colocalized with TGN protein AP1G1. (A) GOLGB1 is dispersed by BFA treatment. MDBK cells were treated with BFA. AP1G1 was detected with AP1G1-specific monoclonal antibody followed by Alexa Fluor 488-conjugated goat anti-mouse IgG. GOLGB1 was detected with GOLGB1-specific polyclonal antibody followed by Alexa Fluor 633-conjugated goat anti-rabbit IgG. (B) VP8 is dispersed by BFA treatment. BoHV-1-infected MDBK cells were treated with BFA. VP8 was detected with VP8-specific polyclonal antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG.

    Techniques Used: Infection

    VP8 causes formation of nuclear lipid droplets in transiently transfected cells. COS-7 cells transfected with different plasmids and Huh-7 cells were fixed for immunostaining. VP8 (WT) was detected with VP8-specific monoclonal antibody followed by Alexa Fluor 488-conjugated goat anti-mouse IgG. US3 was detected with US3-specific polyclonal antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG. Mut-VP8 was detected with VP8-specific polyclonal antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG. Cells were then incubated with Nile red and DAPI.
    Figure Legend Snippet: VP8 causes formation of nuclear lipid droplets in transiently transfected cells. COS-7 cells transfected with different plasmids and Huh-7 cells were fixed for immunostaining. VP8 (WT) was detected with VP8-specific monoclonal antibody followed by Alexa Fluor 488-conjugated goat anti-mouse IgG. US3 was detected with US3-specific polyclonal antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG. Mut-VP8 was detected with VP8-specific polyclonal antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG. Cells were then incubated with Nile red and DAPI.

    Techniques Used: Transfection, Immunostaining, Incubation

    The amount of cytoplasmic FLAG-VP8 increases with the expression level of US3-HA. (A) The intensities of cytoplasmic and nuclear VP8. (B) The intensity of US3 in the nucleus. EBTr cells were transfected with pFLAG-VP8 and incubated for 12 h. This allowed VP8 to be expressed and localized to the nucleus. Subsequently, cells were transfected with pUS3-HA at 12 hpi, 17 hpi, or 22 hpi after transfection with pFLAG-VP8. All samples were fixed at 27 h for immunofluorescent staining, and the cell images were analyzed with the software Leica Application Suite X for protein quantification. VP8 was detected with monoclonal anti-VP8 antibody followed by Alexa Fluor 488-conjugated goat anti-mouse IgG. US3 was identified with polyclonal anti-US3 antibody followed by Alexa Fluor 633-conjugated goat anti-rabbit IgG. The intensities of fluorescence pixels at wavelengths of 488 nm and 633 nm were captured to indicate the quantity of VP8 and US3, respectively. The mean PDU are shown in the bar graphs. Error bars represent the SDs. The statistical significance is shown as follows: *, 0.01
    Figure Legend Snippet: The amount of cytoplasmic FLAG-VP8 increases with the expression level of US3-HA. (A) The intensities of cytoplasmic and nuclear VP8. (B) The intensity of US3 in the nucleus. EBTr cells were transfected with pFLAG-VP8 and incubated for 12 h. This allowed VP8 to be expressed and localized to the nucleus. Subsequently, cells were transfected with pUS3-HA at 12 hpi, 17 hpi, or 22 hpi after transfection with pFLAG-VP8. All samples were fixed at 27 h for immunofluorescent staining, and the cell images were analyzed with the software Leica Application Suite X for protein quantification. VP8 was detected with monoclonal anti-VP8 antibody followed by Alexa Fluor 488-conjugated goat anti-mouse IgG. US3 was identified with polyclonal anti-US3 antibody followed by Alexa Fluor 633-conjugated goat anti-rabbit IgG. The intensities of fluorescence pixels at wavelengths of 488 nm and 633 nm were captured to indicate the quantity of VP8 and US3, respectively. The mean PDU are shown in the bar graphs. Error bars represent the SDs. The statistical significance is shown as follows: *, 0.01

    Techniques Used: Expressing, Transfection, Incubation, Staining, Software, Fluorescence

    Colocalization of VP8 and a cis -Golgi marker protein. MDBK cells mock infected or infected with wild-type BoHV-1 were fixed for immunofluorescent staining. Mock-infected cells shown in the top image were identified with mouse anti-FTCD and rabbit anti-GOLGB1 antibodies. BoHV-1-infected cells in the lower images were identified with mouse anti-VP8 and rabbit anti-GOLGB1 antibodies. Secondary antibodies were Alexa Fluor 488-conjugated goat anti-mouse IgG and Alexa Fluor 633-conjugated goat anti-rabbit IgG, respectively. The cell nuclei were identified with DAPI. Selected areas of the pictures in the first row were zoomed in and are shown on the right side.
    Figure Legend Snippet: Colocalization of VP8 and a cis -Golgi marker protein. MDBK cells mock infected or infected with wild-type BoHV-1 were fixed for immunofluorescent staining. Mock-infected cells shown in the top image were identified with mouse anti-FTCD and rabbit anti-GOLGB1 antibodies. BoHV-1-infected cells in the lower images were identified with mouse anti-VP8 and rabbit anti-GOLGB1 antibodies. Secondary antibodies were Alexa Fluor 488-conjugated goat anti-mouse IgG and Alexa Fluor 633-conjugated goat anti-rabbit IgG, respectively. The cell nuclei were identified with DAPI. Selected areas of the pictures in the first row were zoomed in and are shown on the right side.

    Techniques Used: Marker, Infection, Staining

    6) Product Images from "Recombinant human proteoglycan-4 reduces phagocytosis of urate crystals and downstream nuclear factor kappa B and inflammasome activation and production of cytokines and chemokines in human and murine macrophages"

    Article Title: Recombinant human proteoglycan-4 reduces phagocytosis of urate crystals and downstream nuclear factor kappa B and inflammasome activation and production of cytokines and chemokines in human and murine macrophages

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-018-1693-x

    Colocalization of rhodamine-labeled recombinant human proteoglycan-4 (rhPRG4) (red) and isotype control (IC), CD44 (probed using anti-CD44), toll-like receptor 2 (TLR2) (probed using anti-TLR2) or toll-like receptor 4 (TLR4) (probed using anti-TLR4) in peritoneal Prg4 −/− murine macrophages. Cells were incubated with rhodamine-rhPRG4 for 2 h followed by cell fixation and permeabilization. Following receptor probing, cells were incubated with Alexa Fluor 488 conjugated secondary antibody (green) and counterstained with DAPI (blue). Arrows point to co-localization of rhPRG4 with respective receptors. Quantitative colocalization analysis was performed using Pearson’s Correlation Coefficient and a cutoff of r 2 > 0.5 was used to indicate positive colocalization. The percentage of cells with positive colocalization was determined and at least 100 cells were examined for each treatment condition. Data represent the mean ± S.D. of three independent experiments. Median colocalization images are presented. * p
    Figure Legend Snippet: Colocalization of rhodamine-labeled recombinant human proteoglycan-4 (rhPRG4) (red) and isotype control (IC), CD44 (probed using anti-CD44), toll-like receptor 2 (TLR2) (probed using anti-TLR2) or toll-like receptor 4 (TLR4) (probed using anti-TLR4) in peritoneal Prg4 −/− murine macrophages. Cells were incubated with rhodamine-rhPRG4 for 2 h followed by cell fixation and permeabilization. Following receptor probing, cells were incubated with Alexa Fluor 488 conjugated secondary antibody (green) and counterstained with DAPI (blue). Arrows point to co-localization of rhPRG4 with respective receptors. Quantitative colocalization analysis was performed using Pearson’s Correlation Coefficient and a cutoff of r 2 > 0.5 was used to indicate positive colocalization. The percentage of cells with positive colocalization was determined and at least 100 cells were examined for each treatment condition. Data represent the mean ± S.D. of three independent experiments. Median colocalization images are presented. * p

    Techniques Used: Labeling, Recombinant, Incubation

    7) Product Images from "Tubulin detyrosination promotes human trophoblast syncytium formation"

    Article Title: Tubulin detyrosination promotes human trophoblast syncytium formation

    Journal: Journal of Molecular Cell Biology

    doi: 10.1093/jmcb/mjz084

    Membrane proteins required for fusion are enriched in detyr-α-tub-overexpressing cell membranes. ( A ) Time-lapse images of BeWo cells carrying EGFP-α-tubulin or mCherry-α-tubulin. The cells were treated with forskolin to induce fusion. The white arrowheads indicate where fusion occurred. Scale bar, 10 μm. ( B ) SR-SIM. Alexa Fluor 488 (green) and Alexa Fluor 555 (red) were used to label α-tubulin and detyr-α-tub, respectively, in cells after a 48-h forskolin treatment. Scale bar, 2.5 μm. ( C ) The influences of TTL overexpression or knockdown on the expression of cell surface proteins in different cell fractions of BeWo cells treated with forskolin. The cell surface proteins were isolated and examined with the indicated antibodies. Na + /K + -ATPase served as a plasma membrane loading control. Whole-cell lysates were harvested and examined with the indicated antibodies. GAPDH served as a loading control.
    Figure Legend Snippet: Membrane proteins required for fusion are enriched in detyr-α-tub-overexpressing cell membranes. ( A ) Time-lapse images of BeWo cells carrying EGFP-α-tubulin or mCherry-α-tubulin. The cells were treated with forskolin to induce fusion. The white arrowheads indicate where fusion occurred. Scale bar, 10 μm. ( B ) SR-SIM. Alexa Fluor 488 (green) and Alexa Fluor 555 (red) were used to label α-tubulin and detyr-α-tub, respectively, in cells after a 48-h forskolin treatment. Scale bar, 2.5 μm. ( C ) The influences of TTL overexpression or knockdown on the expression of cell surface proteins in different cell fractions of BeWo cells treated with forskolin. The cell surface proteins were isolated and examined with the indicated antibodies. Na + /K + -ATPase served as a plasma membrane loading control. Whole-cell lysates were harvested and examined with the indicated antibodies. GAPDH served as a loading control.

    Techniques Used: Over Expression, Expressing, Isolation

    Detyrosination of α-tubulin varies during syncytialization of primary CTBs from normal and PE placentae. ( A ) Western blotting of primary CTBs isolated from both normal and PE placentae after spontaneous syncytialization for 72 h using anti-detyr-α-tub, anti-TTL, anti-α-tubulin, anti-Syncytin-2, and anti-GAPDH antibodies. The colored lines represent groups of corresponding gestational ages. ( B ) Quantification of detyr-α-tub from the western blotting data in A . The band intensities detected with anti-detyr-α-tub antibodies were normalized to the band intensity of GAPDH as the loading control. ( C ) The fusion index was determined contemporaneously with the samples harvested for western blotting in A . For confocal microscopy, Alexa Fluor 555 (red) was used to label E-cadherin to identify the cell boundaries. Nuclei were stained with DAPI (blue). The dots and squares representing individual samples from patients are colored according to the matched gestational ages. The horizontal lines represent the means. ( D ) Primary CTBs from preeclamptic placentae undergoing in vitro syncytialization were simultaneously treated with TTL siRNA and subjected to western blotting analysis using anti-detyr-α-tub, anti-TTL, and anti-GAPDH antibodies. The fusion indices were determined as in C . The lines in the enrichment and fusion index results represent groups of PE placentae and normal placentae of corresponding gestational ages. Scale bar, 20 μm.
    Figure Legend Snippet: Detyrosination of α-tubulin varies during syncytialization of primary CTBs from normal and PE placentae. ( A ) Western blotting of primary CTBs isolated from both normal and PE placentae after spontaneous syncytialization for 72 h using anti-detyr-α-tub, anti-TTL, anti-α-tubulin, anti-Syncytin-2, and anti-GAPDH antibodies. The colored lines represent groups of corresponding gestational ages. ( B ) Quantification of detyr-α-tub from the western blotting data in A . The band intensities detected with anti-detyr-α-tub antibodies were normalized to the band intensity of GAPDH as the loading control. ( C ) The fusion index was determined contemporaneously with the samples harvested for western blotting in A . For confocal microscopy, Alexa Fluor 555 (red) was used to label E-cadherin to identify the cell boundaries. Nuclei were stained with DAPI (blue). The dots and squares representing individual samples from patients are colored according to the matched gestational ages. The horizontal lines represent the means. ( D ) Primary CTBs from preeclamptic placentae undergoing in vitro syncytialization were simultaneously treated with TTL siRNA and subjected to western blotting analysis using anti-detyr-α-tub, anti-TTL, and anti-GAPDH antibodies. The fusion indices were determined as in C . The lines in the enrichment and fusion index results represent groups of PE placentae and normal placentae of corresponding gestational ages. Scale bar, 20 μm.

    Techniques Used: Western Blot, Isolation, Confocal Microscopy, Staining, In Vitro

    Augmented detyrosination of α-tubulin during spontaneous fusion of primary CTBs. ( A ) Illustrative model depicting BeWo cell fusion. ( B ) Upper panel: schematic illustration of spontaneous syncytialization of human primary CTBs after isolation. Lower panel: human trophoblasts stained at 0 and 72 h of culture for E-cadherin and nuclei. Scale bar, 50 μm. ( C ) Results of western blotting using the indicated antibodies on primary CTBs from the indicated gestational ages and their corresponding syncytia after 72 h of incubation. GAPDH was used as a loading control (here and hereafter). The colored lines represent groups of corresponding gestational ages. ( D ) Quantification of the western blot data in C . The band intensities detected with anti-α-tubulin and anti-detyr-α-tub antibodies were normalized to that of GAPDH as a control. The dots and squares representing individual samples from patients are colored according to the matching samples at different time points. The horizontal lines represent the means. ( E ) For confocal microscopy, Alexa Fluor 488 (green) was used to label TTL. Alexa Fluor 555 (red) was used to label Syncytin-2, active caspase-8, or CK7. Scale bar, 10 μm. The arrows indicate a layer of mononucleated CTBs and a layer of multinucleated STB.
    Figure Legend Snippet: Augmented detyrosination of α-tubulin during spontaneous fusion of primary CTBs. ( A ) Illustrative model depicting BeWo cell fusion. ( B ) Upper panel: schematic illustration of spontaneous syncytialization of human primary CTBs after isolation. Lower panel: human trophoblasts stained at 0 and 72 h of culture for E-cadherin and nuclei. Scale bar, 50 μm. ( C ) Results of western blotting using the indicated antibodies on primary CTBs from the indicated gestational ages and their corresponding syncytia after 72 h of incubation. GAPDH was used as a loading control (here and hereafter). The colored lines represent groups of corresponding gestational ages. ( D ) Quantification of the western blot data in C . The band intensities detected with anti-α-tubulin and anti-detyr-α-tub antibodies were normalized to that of GAPDH as a control. The dots and squares representing individual samples from patients are colored according to the matching samples at different time points. The horizontal lines represent the means. ( E ) For confocal microscopy, Alexa Fluor 488 (green) was used to label TTL. Alexa Fluor 555 (red) was used to label Syncytin-2, active caspase-8, or CK7. Scale bar, 10 μm. The arrows indicate a layer of mononucleated CTBs and a layer of multinucleated STB.

    Techniques Used: Isolation, Staining, Western Blot, Incubation, Confocal Microscopy

    8) Product Images from "Down-Regulation of Ca2+-Activated K+ Channel KCa1.1 in Human Breast Cancer MDA-MB-453 Cells Treated with Vitamin D Receptor Agonists"

    Article Title: Down-Regulation of Ca2+-Activated K+ Channel KCa1.1 in Human Breast Cancer MDA-MB-453 Cells Treated with Vitamin D Receptor Agonists

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms17122083

    Down-regulation of K Ca 1.1 transcripts and proteins by the treatment with VDR agonists for 72 h in MDA-MB-453 cells. ( A ) Real-time PCR assay for K Ca 1.1 in vehicle-, 1 μM calcitriol-, and 1 μM calcipotriol-treated MDA-MB-453 cells ( n = 4 for each). Expression levels were expressed as a ratio to ACTB; ( B ) Band patterns on agarose gels for the PCR products of K Ca 1.1 exons (exon 1–4, 5–14, 15–23, and 24–30) in vehicle-, 1 µM calcitriol-, and 1 µM calcipotriol-treated MDA-MB-453 cells. A DNA molecular weight marker is indicated on the right of the gel; ( C ) Protein lysates of vehicle-, 1 µM calcitriol-, and 1 µM calcipotriol-treated MDA-MB-453 cells were probed by immunoblotting with anti-K Ca 1.1 (upper panel) and anti-ACTB (lower panel) antibodies on the same filter; ( D ) Summarized results are obtained as the optical density of K Ca 1.1 and ACTB band signals in C . After compensation for the optical density of the K Ca 1.1 protein band signal with that of the ACTB signal, the K Ca 1.1 signal in the vehicle control was expressed as 1.0 (dotted line, n = 3 for each); ( E ) Effects of the treatment with 1 µM calcitriol or 1 µM calcipotriol on the cell surface expression of K Ca 1.1 proteins by a flow cytometric analysis. Non-permeabilized MDA-MB-453 cells were stained with an Alexa Fluor @ 488-conjugated anti-K Ca 1.1 antibody (extracellular). Data were expressed as the relative cell population of K Ca 1.1-positive cells to those in the vehicle control (1.0) ( n = 4 for each). Results are expressed as means ± SEM. ** p
    Figure Legend Snippet: Down-regulation of K Ca 1.1 transcripts and proteins by the treatment with VDR agonists for 72 h in MDA-MB-453 cells. ( A ) Real-time PCR assay for K Ca 1.1 in vehicle-, 1 μM calcitriol-, and 1 μM calcipotriol-treated MDA-MB-453 cells ( n = 4 for each). Expression levels were expressed as a ratio to ACTB; ( B ) Band patterns on agarose gels for the PCR products of K Ca 1.1 exons (exon 1–4, 5–14, 15–23, and 24–30) in vehicle-, 1 µM calcitriol-, and 1 µM calcipotriol-treated MDA-MB-453 cells. A DNA molecular weight marker is indicated on the right of the gel; ( C ) Protein lysates of vehicle-, 1 µM calcitriol-, and 1 µM calcipotriol-treated MDA-MB-453 cells were probed by immunoblotting with anti-K Ca 1.1 (upper panel) and anti-ACTB (lower panel) antibodies on the same filter; ( D ) Summarized results are obtained as the optical density of K Ca 1.1 and ACTB band signals in C . After compensation for the optical density of the K Ca 1.1 protein band signal with that of the ACTB signal, the K Ca 1.1 signal in the vehicle control was expressed as 1.0 (dotted line, n = 3 for each); ( E ) Effects of the treatment with 1 µM calcitriol or 1 µM calcipotriol on the cell surface expression of K Ca 1.1 proteins by a flow cytometric analysis. Non-permeabilized MDA-MB-453 cells were stained with an Alexa Fluor @ 488-conjugated anti-K Ca 1.1 antibody (extracellular). Data were expressed as the relative cell population of K Ca 1.1-positive cells to those in the vehicle control (1.0) ( n = 4 for each). Results are expressed as means ± SEM. ** p

    Techniques Used: Multiple Displacement Amplification, Real-time Polymerase Chain Reaction, Expressing, Polymerase Chain Reaction, Molecular Weight, Marker, Flow Cytometry, Staining

    9) Product Images from "Differential susceptibility of bovine caruncular and trophoblast cell lines to infection with high and low virulence isolates of Neospora caninum"

    Article Title: Differential susceptibility of bovine caruncular and trophoblast cell lines to infection with high and low virulence isolates of Neospora caninum

    Journal: Parasites & Vectors

    doi: 10.1186/s13071-017-2409-9

    Adhesion assay in F3 and BCEC-1 cells infected by Nc-Spain7 and Nc-Spain1H at 4 hpi. Double immunofluorescence staining was performed, and adhered extracellular tachyzoites were stained with Alexa Fluor® 488 ( green ) and Alexa Fluor® 594 ( red ), whereas intracellular tachyzoites were stained with Alexa Fluor® 594 ( red) . Nuclei were stained with DAPI ( blue ). Tachyzoites were counted in 10 arbitrarily selected fields, and the percentage of intracellular tachyzoites relative to the number of total adhered tachyzoites at 4 hpi was calculated. Representative images at a magnification of 1000× ( a ) show the adhesion assay performed in F3 and BCEC-1 cells infected with both isolates. The graph ( b ) represents the percentage of intracellular tachyzoites of Nc-Spain7 and Nc-Spain1H relative to the total number of intra- and extracellular tachyzoites adhered to F3 and BCEC-1 cells. Each column and error bar represents the mean and the SD of 4 replicates from 2 independent assays. BCEC-1 cells showed a significantly higher percentage of intracellular tachyzoites than F3 cells ( P
    Figure Legend Snippet: Adhesion assay in F3 and BCEC-1 cells infected by Nc-Spain7 and Nc-Spain1H at 4 hpi. Double immunofluorescence staining was performed, and adhered extracellular tachyzoites were stained with Alexa Fluor® 488 ( green ) and Alexa Fluor® 594 ( red ), whereas intracellular tachyzoites were stained with Alexa Fluor® 594 ( red) . Nuclei were stained with DAPI ( blue ). Tachyzoites were counted in 10 arbitrarily selected fields, and the percentage of intracellular tachyzoites relative to the number of total adhered tachyzoites at 4 hpi was calculated. Representative images at a magnification of 1000× ( a ) show the adhesion assay performed in F3 and BCEC-1 cells infected with both isolates. The graph ( b ) represents the percentage of intracellular tachyzoites of Nc-Spain7 and Nc-Spain1H relative to the total number of intra- and extracellular tachyzoites adhered to F3 and BCEC-1 cells. Each column and error bar represents the mean and the SD of 4 replicates from 2 independent assays. BCEC-1 cells showed a significantly higher percentage of intracellular tachyzoites than F3 cells ( P

    Techniques Used: Cell Adhesion Assay, Infection, Double Immunofluorescence Staining, Staining

    10) Product Images from "Extracellular annexins and dynamin are important for sequential steps in myoblast fusion"

    Article Title: Extracellular annexins and dynamin are important for sequential steps in myoblast fusion

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201207012

    Synchronized fusion of C2C12 cells is influenced by reagents targeting extracellular Anx A1 and A5. (A) Phase-contrast images with nuclear staining (blue) showing C2C12 cells that were incubated in DM for 51 h and then in LPC-supplemented DM for 16 h, and, finally, placed into LPC-free DM (left) or LPC-free DM with antibodies to Anx A1 or A5 (middle and right). The images were taken 30 min after LPC removal. Arrows mark the multinucleated cells. Bar, 50 µm. (B–D) Antibodies (B), A1- and A5-peptides (C), and EGTA (D) were applied to ready-to-fuse myoblasts at the time of LPC removal (B and C) or 30 min before LPC removal (D). Fusion was scored 30 (B and C) or 60 (D) min after LPC removal and normalized to fusion in the control experiments shown (B–D, 1). (B) Lipid mixing (green) and syncytium formation (red) were inhibited by antibodies to Anx A1 (2) and A5 (3) but not by nonspecific IgG (4). 1, untreated cells released from LPC block. (C) Fusion was inhibited by the peptides to Anx A1 (2) and A5 (4) but not by their scrambled versions (3 and 5). (D) At the time of LPC removal, the cells were placed into Ca 2+ - and Mg 2+ -free LPC-free PBS supplemented with 10 mM EGTA (2–7). (3–5) 30 min after LPC removal, we washed the cells with EGTA-free Ca 2+ - and Mg 2+ -containing PBS (3) or with the same buffer supplemented with rA1 (4) or rA5 (5). (6 and 7) As in 4 and 5, but rA1 (6) and rA5 (7) were applied in Ca 2+ - and Mg 2+ -free PBS. (1) At the time of LPC removal, the cells were placed into Ca 2+ - and Mg 2+ -containing PBS. All results are means ± SEM ( n ≥ 3). (B–D) Levels of significance relative to 1 in B and C and to 3 in D are shown: **, P
    Figure Legend Snippet: Synchronized fusion of C2C12 cells is influenced by reagents targeting extracellular Anx A1 and A5. (A) Phase-contrast images with nuclear staining (blue) showing C2C12 cells that were incubated in DM for 51 h and then in LPC-supplemented DM for 16 h, and, finally, placed into LPC-free DM (left) or LPC-free DM with antibodies to Anx A1 or A5 (middle and right). The images were taken 30 min after LPC removal. Arrows mark the multinucleated cells. Bar, 50 µm. (B–D) Antibodies (B), A1- and A5-peptides (C), and EGTA (D) were applied to ready-to-fuse myoblasts at the time of LPC removal (B and C) or 30 min before LPC removal (D). Fusion was scored 30 (B and C) or 60 (D) min after LPC removal and normalized to fusion in the control experiments shown (B–D, 1). (B) Lipid mixing (green) and syncytium formation (red) were inhibited by antibodies to Anx A1 (2) and A5 (3) but not by nonspecific IgG (4). 1, untreated cells released from LPC block. (C) Fusion was inhibited by the peptides to Anx A1 (2) and A5 (4) but not by their scrambled versions (3 and 5). (D) At the time of LPC removal, the cells were placed into Ca 2+ - and Mg 2+ -free LPC-free PBS supplemented with 10 mM EGTA (2–7). (3–5) 30 min after LPC removal, we washed the cells with EGTA-free Ca 2+ - and Mg 2+ -containing PBS (3) or with the same buffer supplemented with rA1 (4) or rA5 (5). (6 and 7) As in 4 and 5, but rA1 (6) and rA5 (7) were applied in Ca 2+ - and Mg 2+ -free PBS. (1) At the time of LPC removal, the cells were placed into Ca 2+ - and Mg 2+ -containing PBS. All results are means ± SEM ( n ≥ 3). (B–D) Levels of significance relative to 1 in B and C and to 3 in D are shown: **, P

    Techniques Used: Staining, Incubation, Blocking Assay

    11) Product Images from "Hippocampal clock regulates memory retrieval via Dopamine and PKA-induced GluA1 phosphorylation"

    Article Title: Hippocampal clock regulates memory retrieval via Dopamine and PKA-induced GluA1 phosphorylation

    Journal: Nature Communications

    doi: 10.1038/s41467-019-13554-y

    Forebrain-specific inhibition of BMAL1 function. a dnBMAL1 mRNA expression in dnBMAL1 mice. Dox-dependent dnBMAL1 mRNA expression in hippocampus (RT-PCR, left). dnBMAL1 mRNA expression in hippocampus (HPC, middle) but not SCN (right) (in situ hybridization). AVP mRNA expression as a marker of SCN. DAPI (nuclear stain, blue), dnBMAL1 (green), AVP (red). Scale bar, 200 μm (HPC) and 100 μm (SCN). b , c PER2 and expression levels (BMAL1 target genes) are reduced in hippocampal CA1 ( b ) but not in SCN ( c ), in dnBMAL1 mice at both ZT4 and 10. The graph represents fold changes compared to the expression levels in WT at ZT4. d dnBMAL1 blocks the CLOCK binding to Dbp promoter in the hippocampus of dnBMAL1 mice at ZT10. Anti-CLOCK antibody, but not anti-IgG, precipitated DBP promoter although DNA regions not containing E-box ( clock gene exon 6) are comparably precipitated by anti-CLOCK antibody and anti-IgG. e Normal circadian locomotor rhythm in dnBMAL1 mice. Mice were housed in a 12 h light:12 h dark (LD) cycle then in constant darkness (DD). (Left) Representative activity records are double-plotted with each horizontal line representing 48 h. Circadian period (Middle) and daily locomotor activity (Right) under DD. All values are mean ± SEM. Individual data points are displayed as dots. * p
    Figure Legend Snippet: Forebrain-specific inhibition of BMAL1 function. a dnBMAL1 mRNA expression in dnBMAL1 mice. Dox-dependent dnBMAL1 mRNA expression in hippocampus (RT-PCR, left). dnBMAL1 mRNA expression in hippocampus (HPC, middle) but not SCN (right) (in situ hybridization). AVP mRNA expression as a marker of SCN. DAPI (nuclear stain, blue), dnBMAL1 (green), AVP (red). Scale bar, 200 μm (HPC) and 100 μm (SCN). b , c PER2 and expression levels (BMAL1 target genes) are reduced in hippocampal CA1 ( b ) but not in SCN ( c ), in dnBMAL1 mice at both ZT4 and 10. The graph represents fold changes compared to the expression levels in WT at ZT4. d dnBMAL1 blocks the CLOCK binding to Dbp promoter in the hippocampus of dnBMAL1 mice at ZT10. Anti-CLOCK antibody, but not anti-IgG, precipitated DBP promoter although DNA regions not containing E-box ( clock gene exon 6) are comparably precipitated by anti-CLOCK antibody and anti-IgG. e Normal circadian locomotor rhythm in dnBMAL1 mice. Mice were housed in a 12 h light:12 h dark (LD) cycle then in constant darkness (DD). (Left) Representative activity records are double-plotted with each horizontal line representing 48 h. Circadian period (Middle) and daily locomotor activity (Right) under DD. All values are mean ± SEM. Individual data points are displayed as dots. * p

    Techniques Used: Inhibition, Expressing, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, In Situ Hybridization, Marker, Staining, Binding Assay, Activity Assay

    12) Product Images from "A Stem-Cell-Derived Platform Enables Complete Cryptosporidium Development In Vitro and Genetic Tractability"

    Article Title: A Stem-Cell-Derived Platform Enables Complete Cryptosporidium Development In Vitro and Genetic Tractability

    Journal: Cell Host & Microbe

    doi: 10.1016/j.chom.2019.05.007

    In vitro production of oocysts in ALI culture (A) Effect of filtration on removal of residual C. parvum oocysts. Oocysts were excysted, filtered using the indicated pore sizes, added to PLL-coated coverslips, and stained with Crypt-a-glo directly conjugated to FITC. The number of residual oocysts found in filtered samples were counted from replicate 63× fields from a representative experiment. (B) Detection of C. parvum oocysts in ALI cultures. Transwells were infected on day 3 post top medium removal with 1 μm-filtered sporozoites. On specified days post infection, transwells were fixed and stained with Crypt-a-glo directly conjugated to FITC and Pan Cp, detected with goat anti-rabbit IgG Alexa Fluor 568. Oocyst numbers per 63× field were counted for three independent experiments (Exp A, Exp B, Exp C). The combined mean of each time point was compared to the combined mean of day 1 using a two-way ANOVA corrected for multiple comparisons using the Dunnett method, ∗∗∗∗ p
    Figure Legend Snippet: In vitro production of oocysts in ALI culture (A) Effect of filtration on removal of residual C. parvum oocysts. Oocysts were excysted, filtered using the indicated pore sizes, added to PLL-coated coverslips, and stained with Crypt-a-glo directly conjugated to FITC. The number of residual oocysts found in filtered samples were counted from replicate 63× fields from a representative experiment. (B) Detection of C. parvum oocysts in ALI cultures. Transwells were infected on day 3 post top medium removal with 1 μm-filtered sporozoites. On specified days post infection, transwells were fixed and stained with Crypt-a-glo directly conjugated to FITC and Pan Cp, detected with goat anti-rabbit IgG Alexa Fluor 568. Oocyst numbers per 63× field were counted for three independent experiments (Exp A, Exp B, Exp C). The combined mean of each time point was compared to the combined mean of day 1 using a two-way ANOVA corrected for multiple comparisons using the Dunnett method, ∗∗∗∗ p

    Techniques Used: In Vitro, Filtration, Staining, Infection

    ALI Transwells support genetic crosses of C. parvum in vitro (A) Diagram of targeting construct designed to replace the endogenous tk locus (cgd5_4440) with GFP and Nluc-P2A-Neo R cassette. (B) Relative luminescence normalized to total number of parasites in ALI transwells at 1 and 4 days post infection (dpi). Transwells were infected with ∼1 × 10 7 unfiltered sporozoites that were electroporated with the TK-GFP-Nluc-P2A-neo-TK reporter and a Cas9 plasmid with a TK gRNA. Transwells were cultured in medium containing PBS (light green) as a control or 20 mM paromomycin (PRM, dark green). Data plotted as mean ± S.D. from two transwells per time point from a representative experiment. Nonsignificant (p = 0.11), unpaired Student’s t test between PBS and PRM-treated transwells 4 dpi. (C) Image of whole-mount ALI transwells 5 dpi with transfected C. parvum from same experiment as (B) stained with anti-GFP followed by goat anti-rabbit IgG Alexa Fluor 488. Scale bar, 10 μm. (D) Merged image of (C) with a Pan Cp polyclonal antibody, which recognizes all C. parvum stages, followed by goat antirat IgG Alexa Fluor 568. Scale bar, 10 μm. (E) Diagram of targeting construct designed to replace the endogenous uprt locus (cgd1_1900) with mCherry and Nluc-P2A-Neo R cassette as (A). (F) Relative luminescence normalized to total number of parasites in ALI transwells at 1 and 3 dpi. Transwells were infected with ∼1 × 10 7 unfiltered sporozoites per transwell that were electroporated with the UPRT-mCh-Nluc-P2A-neo-UPRT reporter and a Cas9 plasmid with a UPRT gRNA. Transwells were cultured in medium containing PBS (pink) as a control or 20 mM PRM (red). Data plotted as mean ± S.D. from two transwells per time point from a representative experiment. ∗ p
    Figure Legend Snippet: ALI Transwells support genetic crosses of C. parvum in vitro (A) Diagram of targeting construct designed to replace the endogenous tk locus (cgd5_4440) with GFP and Nluc-P2A-Neo R cassette. (B) Relative luminescence normalized to total number of parasites in ALI transwells at 1 and 4 days post infection (dpi). Transwells were infected with ∼1 × 10 7 unfiltered sporozoites that were electroporated with the TK-GFP-Nluc-P2A-neo-TK reporter and a Cas9 plasmid with a TK gRNA. Transwells were cultured in medium containing PBS (light green) as a control or 20 mM paromomycin (PRM, dark green). Data plotted as mean ± S.D. from two transwells per time point from a representative experiment. Nonsignificant (p = 0.11), unpaired Student’s t test between PBS and PRM-treated transwells 4 dpi. (C) Image of whole-mount ALI transwells 5 dpi with transfected C. parvum from same experiment as (B) stained with anti-GFP followed by goat anti-rabbit IgG Alexa Fluor 488. Scale bar, 10 μm. (D) Merged image of (C) with a Pan Cp polyclonal antibody, which recognizes all C. parvum stages, followed by goat antirat IgG Alexa Fluor 568. Scale bar, 10 μm. (E) Diagram of targeting construct designed to replace the endogenous uprt locus (cgd1_1900) with mCherry and Nluc-P2A-Neo R cassette as (A). (F) Relative luminescence normalized to total number of parasites in ALI transwells at 1 and 3 dpi. Transwells were infected with ∼1 × 10 7 unfiltered sporozoites per transwell that were electroporated with the UPRT-mCh-Nluc-P2A-neo-UPRT reporter and a Cas9 plasmid with a UPRT gRNA. Transwells were cultured in medium containing PBS (pink) as a control or 20 mM PRM (red). Data plotted as mean ± S.D. from two transwells per time point from a representative experiment. ∗ p

    Techniques Used: In Vitro, Construct, Infection, Plasmid Preparation, Cell Culture, Transfection, Staining

    Development of an In Vitro System for Cultivation of C. Parvum (A) Model of air-liquid interface (ALI) culture method. See methods for details and Figures S1 A and S1B for further description. (B) Histological examination of ALI cultures. Sections of ALI 3 days post-infection (PI) stained with hematoxylin and eosin (H E), or rabbit pAb to detect C. parvum (referred to as Pan Cp) using immunohistochemistry (IHC). White arrows in middle panel highlight C. parvum . Scale bar, 20 μm. (C) Growth of C. parvum in ALI cultures infected 3 days post top medium removal with 2 × 10 5 unfiltered C. parvum oocysts. The graph depicts qPCR measurement of C. parvum and mouse GAPDH equivalents (eq). Means ± S.D. of two transwells per time point from a representative experiment. See Figure S1 C for the replicate experiment. (D) Detection of developmental stages of C. parvum in ALI cultures. Infected ALI transwells were fixed and stained with specified mouse mAbs (i.e., 1B5, 1A5) followed by goat antimouse IgG Alexa Fluor 488, Crypt-a-glo directly conjugated to FITC, or Pan Cp followed by goat anti-rabbit IgG Alexa Fluor 568. Hoechst staining for DNA. Scale bar, 3 μm. See also Figure S2 .
    Figure Legend Snippet: Development of an In Vitro System for Cultivation of C. Parvum (A) Model of air-liquid interface (ALI) culture method. See methods for details and Figures S1 A and S1B for further description. (B) Histological examination of ALI cultures. Sections of ALI 3 days post-infection (PI) stained with hematoxylin and eosin (H E), or rabbit pAb to detect C. parvum (referred to as Pan Cp) using immunohistochemistry (IHC). White arrows in middle panel highlight C. parvum . Scale bar, 20 μm. (C) Growth of C. parvum in ALI cultures infected 3 days post top medium removal with 2 × 10 5 unfiltered C. parvum oocysts. The graph depicts qPCR measurement of C. parvum and mouse GAPDH equivalents (eq). Means ± S.D. of two transwells per time point from a representative experiment. See Figure S1 C for the replicate experiment. (D) Detection of developmental stages of C. parvum in ALI cultures. Infected ALI transwells were fixed and stained with specified mouse mAbs (i.e., 1B5, 1A5) followed by goat antimouse IgG Alexa Fluor 488, Crypt-a-glo directly conjugated to FITC, or Pan Cp followed by goat anti-rabbit IgG Alexa Fluor 568. Hoechst staining for DNA. Scale bar, 3 μm. See also Figure S2 .

    Techniques Used: In Vitro, Infection, Staining, Immunohistochemistry, Real-time Polymerase Chain Reaction

    13) Product Images from "Rev-erb agonist improves adverse cardiac remodeling and survival in myocardial infarction through an anti-inflammatory mechanism"

    Article Title: Rev-erb agonist improves adverse cardiac remodeling and survival in myocardial infarction through an anti-inflammatory mechanism

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0189330

    Representative immunofluorescence images of the left ventricles stained with MMP-9. (A) Sham+V, (B) Sham+SR, (C) MI+V, and (D) MI+SR. MMP-9 was stained with Alexa Fluor 594 (red) and nuclei were stained with DAPI (blue). All images show 40× magnification. Sham groups showed low immunofluorescence intensity, and MMP-9 was strongly detected in the infarct and border areas in the MI groups. MI+SR showed less intensity compared with MI+V. (E) The ratio of immunofluorescence-positive area to total area was significantly increased in MI+V, and was markedly decreased in MI+SR (n = 4 animals in each group). The bar graphs show the group mean±SEM. *p
    Figure Legend Snippet: Representative immunofluorescence images of the left ventricles stained with MMP-9. (A) Sham+V, (B) Sham+SR, (C) MI+V, and (D) MI+SR. MMP-9 was stained with Alexa Fluor 594 (red) and nuclei were stained with DAPI (blue). All images show 40× magnification. Sham groups showed low immunofluorescence intensity, and MMP-9 was strongly detected in the infarct and border areas in the MI groups. MI+SR showed less intensity compared with MI+V. (E) The ratio of immunofluorescence-positive area to total area was significantly increased in MI+V, and was markedly decreased in MI+SR (n = 4 animals in each group). The bar graphs show the group mean±SEM. *p

    Techniques Used: Immunofluorescence, Staining

    14) Product Images from "NOD2-mediated Suppression of CD55 on Neutrophils Enhances C5a Generation During Polymicrobial Sepsis"

    Article Title: NOD2-mediated Suppression of CD55 on Neutrophils Enhances C5a Generation During Polymicrobial Sepsis

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003351

    SB203580, an RIP2 inhibitor downstream of nucleotide-binding oligomerization domain (NOD)2, attenuates CLP-induced sepsis. (A) Peritoneal cells of WT mice were cultured with SB203580 and/or MDP for 24 h, and IL-1β and IL-10 concentrations were measured in culture fractions. (B) Molecules related to NOD2-mediated signal transduction were blotted using peritoneal cells obtained from WT and Nod2 −/− mice injected with SB203580 or PBS 24 h after CLP. (C) Serum and peritoneal IL-1β, IL-10, and C5a levels were estimated in WT (n = 4) and Nod2 −/− (n = 3) mice injected with SB203580 (n = 4 in WT, n = 3 in Nod2 −/− ) or PBS 24 h after CLP by ELISA. (D) The levels of CD55 expression on F4/80 − Ly6-G + cells from WT (n = 3) and WT mice injected with SB203580 (n = 3) were measured 24 h after CLP. (mean fluorescence intensity [MFI] of CD55 expression in the panels, diagrams filled with gray for istotype-matched control IgG, solid lines for CD55) (E) The percentages of surviving mice were estimated during CLP-induced sepsis ( a P = 0.0312, log-rank test, n = 6–8 per group; WT mice injected with SB203580 vs. PBS). *P
    Figure Legend Snippet: SB203580, an RIP2 inhibitor downstream of nucleotide-binding oligomerization domain (NOD)2, attenuates CLP-induced sepsis. (A) Peritoneal cells of WT mice were cultured with SB203580 and/or MDP for 24 h, and IL-1β and IL-10 concentrations were measured in culture fractions. (B) Molecules related to NOD2-mediated signal transduction were blotted using peritoneal cells obtained from WT and Nod2 −/− mice injected with SB203580 or PBS 24 h after CLP. (C) Serum and peritoneal IL-1β, IL-10, and C5a levels were estimated in WT (n = 4) and Nod2 −/− (n = 3) mice injected with SB203580 (n = 4 in WT, n = 3 in Nod2 −/− ) or PBS 24 h after CLP by ELISA. (D) The levels of CD55 expression on F4/80 − Ly6-G + cells from WT (n = 3) and WT mice injected with SB203580 (n = 3) were measured 24 h after CLP. (mean fluorescence intensity [MFI] of CD55 expression in the panels, diagrams filled with gray for istotype-matched control IgG, solid lines for CD55) (E) The percentages of surviving mice were estimated during CLP-induced sepsis ( a P = 0.0312, log-rank test, n = 6–8 per group; WT mice injected with SB203580 vs. PBS). *P

    Techniques Used: Binding Assay, Mouse Assay, Cell Culture, Transduction, Injection, Enzyme-linked Immunosorbent Assay, Expressing, Fluorescence

    IL-1β-dependent IL-10 production mediated by nucleotide-binding oligomerization domain (NOD) 2 enhances C5a generation by suppressing CD55 expression on Ly6-G + cells during sepsis. (A) CD55 and CR1/2 expression on gated F4/80 − Ly6-G + peritoneal cells from WT, Nod2 −/− , and Nod2 −/− mice injected with recombinant IL-1β or IL-10 was estimated 24 h after CLP (mean fluorescence intensity [MFI] of CD55 expression in the panels). (B) CD55 expression on gated F4/80 − Ly6-G + peritoneal cells from Il-10 −/− or Il-10 −/− mice injected with recombinant IL-1β was estimated 24 h after CLP (MFI of CD55 expression in the panels) (C) To block IL-10 receptor engagement in vivo , anti-IL10 receptor mAbs were i.p. injected into WT and Nod2 −/− mice administered recombinant IL-10 during CLP-induced sepsis. CD55 expression on gated F4/80 − Ly6-G + peritoneal cells from these mice 24 h after CLP was evaluated. (D) The levels of CD55 expression on F4/80 − Ly6-G + peritoneal cells were compared in WT, Nod2 −/− , IL-10 −/− , and Il-1r −/− mice 24 h after CLP. (A–D) anti-CD55 mAb (lines) and control IgG (diagrams filled with gray) were used. (E) Peritoneal cells from WT and Nod2 −/− mice 24 h after CLP were blotted for Bb factor. (F) Peritoneal cells from WT and Nod2 −/− mice 12 h after CLP were incubated with RPMI media containing 10% WT mouse serum for 24 h. (G and H) To evaluate the effect of CD55 on C5a generation in vivo , WT, Nod2 −/− , (G) or Nod2 −/− mice given recombinant IL-10 (H) were i.p. injected with soluble CD55 12 h after CLP. Serum and peritoneal C5a levels and the survival percentages of these mice were measured during CLP-induced sepsis ( a P = 0.0124, log-rank test; WT [n = 8], Nod2 −/− [n = 8 ], and soluble CD55-injected WT [n = 6 ] or Nod2 −/− mice [n = 8 ] in G, a P = 0.0024, log-rank test; Nod2 −/− mice [n = 8], Nod2 −/− mice injected with recombinant IL-10 [n = 8] or recombinant IL-10 and soluble CD55 [n = 6] in H). *P
    Figure Legend Snippet: IL-1β-dependent IL-10 production mediated by nucleotide-binding oligomerization domain (NOD) 2 enhances C5a generation by suppressing CD55 expression on Ly6-G + cells during sepsis. (A) CD55 and CR1/2 expression on gated F4/80 − Ly6-G + peritoneal cells from WT, Nod2 −/− , and Nod2 −/− mice injected with recombinant IL-1β or IL-10 was estimated 24 h after CLP (mean fluorescence intensity [MFI] of CD55 expression in the panels). (B) CD55 expression on gated F4/80 − Ly6-G + peritoneal cells from Il-10 −/− or Il-10 −/− mice injected with recombinant IL-1β was estimated 24 h after CLP (MFI of CD55 expression in the panels) (C) To block IL-10 receptor engagement in vivo , anti-IL10 receptor mAbs were i.p. injected into WT and Nod2 −/− mice administered recombinant IL-10 during CLP-induced sepsis. CD55 expression on gated F4/80 − Ly6-G + peritoneal cells from these mice 24 h after CLP was evaluated. (D) The levels of CD55 expression on F4/80 − Ly6-G + peritoneal cells were compared in WT, Nod2 −/− , IL-10 −/− , and Il-1r −/− mice 24 h after CLP. (A–D) anti-CD55 mAb (lines) and control IgG (diagrams filled with gray) were used. (E) Peritoneal cells from WT and Nod2 −/− mice 24 h after CLP were blotted for Bb factor. (F) Peritoneal cells from WT and Nod2 −/− mice 12 h after CLP were incubated with RPMI media containing 10% WT mouse serum for 24 h. (G and H) To evaluate the effect of CD55 on C5a generation in vivo , WT, Nod2 −/− , (G) or Nod2 −/− mice given recombinant IL-10 (H) were i.p. injected with soluble CD55 12 h after CLP. Serum and peritoneal C5a levels and the survival percentages of these mice were measured during CLP-induced sepsis ( a P = 0.0124, log-rank test; WT [n = 8], Nod2 −/− [n = 8 ], and soluble CD55-injected WT [n = 6 ] or Nod2 −/− mice [n = 8 ] in G, a P = 0.0024, log-rank test; Nod2 −/− mice [n = 8], Nod2 −/− mice injected with recombinant IL-10 [n = 8] or recombinant IL-10 and soluble CD55 [n = 6] in H). *P

    Techniques Used: Binding Assay, Expressing, Mouse Assay, Injection, Recombinant, Fluorescence, Blocking Assay, In Vivo, Incubation

    15) Product Images from "Impact of angiotensin-converting enzyme inhibition on platelet tissue factor expression in stroke-prone rats"

    Article Title: Impact of angiotensin-converting enzyme inhibition on platelet tissue factor expression in stroke-prone rats

    Journal: Journal of Hypertension

    doi: 10.1097/HJH.0000000000001702

    Flow cytometry analysis of cell-surface tissue factor expression in rat and human platelets stimulated with angiotensin II. Platelet-rich plasma from normotensive Wistar Kyoto rats was stimulated with increasing concentration of collagen (a) or, a selective AT1 agonist (b), and stained with polyclonal antitissue factor antibody labeled with Alexa Fluor 633. The effect of AngII on human platelets is also reported (c). Mean percentage of tissue factor-positive cells ± SD, in each experimental setting, is reported as histogram. ANOVA was applied for statistical analysis. P values were obtained by post-hoc test.
    Figure Legend Snippet: Flow cytometry analysis of cell-surface tissue factor expression in rat and human platelets stimulated with angiotensin II. Platelet-rich plasma from normotensive Wistar Kyoto rats was stimulated with increasing concentration of collagen (a) or, a selective AT1 agonist (b), and stained with polyclonal antitissue factor antibody labeled with Alexa Fluor 633. The effect of AngII on human platelets is also reported (c). Mean percentage of tissue factor-positive cells ± SD, in each experimental setting, is reported as histogram. ANOVA was applied for statistical analysis. P values were obtained by post-hoc test.

    Techniques Used: Flow Cytometry, Cytometry, Expressing, Concentration Assay, Staining, Labeling

    16) Product Images from "Gold nanocage-based lateral flow immunoassay for immunoglobulin G"

    Article Title: Gold nanocage-based lateral flow immunoassay for immunoglobulin G

    Journal: Mikrochimica acta

    doi: 10.1007/s00604-017-2176-5

    Typical recorded responses of LFSBs with a portable strip reader after applying the sample solutions (right: optical response of the control line; left: optical response of the test line). 0 ng mL −1 ( a ), 0.5 ng mL −1 ( b ), 5 ng mL −1 ( c ), 50 ng mL −1 ( d ) of IgG. Assay time: 15 min; all sample solution were prepared with PBST buffer
    Figure Legend Snippet: Typical recorded responses of LFSBs with a portable strip reader after applying the sample solutions (right: optical response of the control line; left: optical response of the test line). 0 ng mL −1 ( a ), 0.5 ng mL −1 ( b ), 5 ng mL −1 ( c ), 50 ng mL −1 ( d ) of IgG. Assay time: 15 min; all sample solution were prepared with PBST buffer

    Techniques Used: Stripping Membranes

    17) Product Images from "Stromal mesenchymal stem cells facilitate pancreatic cancer progression by regulating specific secretory molecules through mutual cellular interaction"

    Article Title: Stromal mesenchymal stem cells facilitate pancreatic cancer progression by regulating specific secretory molecules through mutual cellular interaction

    Journal: Journal of Cancer

    doi: 10.7150/jca.24415

    Immunophenotypic characterization of human MSC and MSC-mediated increase in invasiveness of pancreatic cancer cells. A. FACS analysis of human MSC using anti-human CD34, CD45, CD73, and CD105. Mouse IgG as classmatch control for mouse monoclonal CD34, CD45, rabbit IgG for rabbit monoclonal CD73 and CD105. Indirect immunofluorescence using Alexa488-conjugated secondary antibodies. B and C. BxPC3 invasiveness was assessed under the co-culture condition with hMSCs by employing Boyden chamber method. Filter membrane set in the top chamber was sealed with Matrigel to close the micro pore. The mixed cells (BxPC3-GFP cells and hMSCs, ratio 1:1) were placed in the top chamber filled with serum free medium and serum was added or not added to the bottom well to be final concentrations 0.5% and 10%. After incubation for 12 hours, passed BxPC3-GFP cells through the filter membrane were observed by the fluorescence microscope (A) and their fluorescence intensities were measured and quantified (B). Data from A are means ± SD, *P
    Figure Legend Snippet: Immunophenotypic characterization of human MSC and MSC-mediated increase in invasiveness of pancreatic cancer cells. A. FACS analysis of human MSC using anti-human CD34, CD45, CD73, and CD105. Mouse IgG as classmatch control for mouse monoclonal CD34, CD45, rabbit IgG for rabbit monoclonal CD73 and CD105. Indirect immunofluorescence using Alexa488-conjugated secondary antibodies. B and C. BxPC3 invasiveness was assessed under the co-culture condition with hMSCs by employing Boyden chamber method. Filter membrane set in the top chamber was sealed with Matrigel to close the micro pore. The mixed cells (BxPC3-GFP cells and hMSCs, ratio 1:1) were placed in the top chamber filled with serum free medium and serum was added or not added to the bottom well to be final concentrations 0.5% and 10%. After incubation for 12 hours, passed BxPC3-GFP cells through the filter membrane were observed by the fluorescence microscope (A) and their fluorescence intensities were measured and quantified (B). Data from A are means ± SD, *P

    Techniques Used: FACS, Immunofluorescence, Co-Culture Assay, Incubation, Fluorescence, Microscopy

    18) Product Images from "Identification of specific biomarkers for gastric adenocarcinoma by ITRAQ proteomic approach"

    Article Title: Identification of specific biomarkers for gastric adenocarcinoma by ITRAQ proteomic approach

    Journal: Scientific Reports

    doi: 10.1038/srep38871

    Validation of UQCRC1 expression by immunofluorescence staining. Frozen sections from stage I and III GC ( A ) and their adjacent normal gastric tissues ( B ) were incubated with antibody against human UQCRC1, followed by goat anti-rabbit IgG–Alexa Fluor® 568 conjugate. Nuclei are stained with DAPI (blue).
    Figure Legend Snippet: Validation of UQCRC1 expression by immunofluorescence staining. Frozen sections from stage I and III GC ( A ) and their adjacent normal gastric tissues ( B ) were incubated with antibody against human UQCRC1, followed by goat anti-rabbit IgG–Alexa Fluor® 568 conjugate. Nuclei are stained with DAPI (blue).

    Techniques Used: Expressing, Immunofluorescence, Staining, Incubation

    Validation of Annexin A1 expression by immunofluorescence staining. Frozen sections from stage I and III GC ( A ) and their adjacent normal gastric tissues ( B ) were incubated with antibody against human Annexin A1, followed by goat anti-rabbit IgG - Alexa Fluor® 568 conjugate. Nuclei are stained with DAPI (blue).
    Figure Legend Snippet: Validation of Annexin A1 expression by immunofluorescence staining. Frozen sections from stage I and III GC ( A ) and their adjacent normal gastric tissues ( B ) were incubated with antibody against human Annexin A1, followed by goat anti-rabbit IgG - Alexa Fluor® 568 conjugate. Nuclei are stained with DAPI (blue).

    Techniques Used: Expressing, Immunofluorescence, Staining, Incubation

    19) Product Images from "Characterization of a midgut mucin-like glycoconjugate of Lutzomyia longipalpis with a potential role in Leishmania attachment"

    Article Title: Characterization of a midgut mucin-like glycoconjugate of Lutzomyia longipalpis with a potential role in Leishmania attachment

    Journal: Parasites & Vectors

    doi: 10.1186/s13071-016-1695-y

    Binding of recombinant Lulo protein to Leishmania major promastigotes. Promastigotes were fixed on slides and incubated with recombinant Lulo protein, followed by anti-rLulo serum incubation and Alexa-anti rabbit IgG labelling. The entire body, including the flagellum was brightly stained by green fluorescent staining ( a ). In contrast, parasites incubated with preimunne serum instead of anti-rLulo serum were not stained with Alexa-anti rabbit IgG ( b ). In the negative controls, parasites incubated with BSA instead of rLulo protein, then with anti-rLulo serum and followed by Alexa staining were stained only weakly ( c ) and parasites incubated only with Alexa fluor labelling were not stained at all ( d )
    Figure Legend Snippet: Binding of recombinant Lulo protein to Leishmania major promastigotes. Promastigotes were fixed on slides and incubated with recombinant Lulo protein, followed by anti-rLulo serum incubation and Alexa-anti rabbit IgG labelling. The entire body, including the flagellum was brightly stained by green fluorescent staining ( a ). In contrast, parasites incubated with preimunne serum instead of anti-rLulo serum were not stained with Alexa-anti rabbit IgG ( b ). In the negative controls, parasites incubated with BSA instead of rLulo protein, then with anti-rLulo serum and followed by Alexa staining were stained only weakly ( c ) and parasites incubated only with Alexa fluor labelling were not stained at all ( d )

    Techniques Used: Binding Assay, Recombinant, Incubation, Staining

    20) Product Images from "Prolonged Pharmacokinetic and Pharmacodynamic Actions of a Pegylated Parathyroid Hormone (1-34) Peptide Fragment"

    Article Title: Prolonged Pharmacokinetic and Pharmacodynamic Actions of a Pegylated Parathyroid Hormone (1-34) Peptide Fragment

    Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

    doi: 10.1002/jbmr.2917

    Confocal microscopy analysis of TMR-peptide and phosphorylated PKA substrate in kidney. Confocal microscopy images of sections of renal cortex derived from mice at 5 minutes, 60 minutes, or 24 hours after intravenous injection with either a nonspecific control peptide, NS TMR ( A ), PTH TMR ( BE ), or PEG-PTH TMR ( F, G ). Sections were immunostained (green) using a primary IgG antibody directed against phosphorylated PKA substrates and a secondary anti-rabbit IgG antibody labeled with Fluor-488. Sections were imaged for pKA-substrate (green), TMR-peptide (red), and DAPI (blue) at a total magnification of 600× or 1800× ( C, E ). No immunostaining was detected in sections not treated with primary antibody.
    Figure Legend Snippet: Confocal microscopy analysis of TMR-peptide and phosphorylated PKA substrate in kidney. Confocal microscopy images of sections of renal cortex derived from mice at 5 minutes, 60 minutes, or 24 hours after intravenous injection with either a nonspecific control peptide, NS TMR ( A ), PTH TMR ( BE ), or PEG-PTH TMR ( F, G ). Sections were immunostained (green) using a primary IgG antibody directed against phosphorylated PKA substrates and a secondary anti-rabbit IgG antibody labeled with Fluor-488. Sections were imaged for pKA-substrate (green), TMR-peptide (red), and DAPI (blue) at a total magnification of 600× or 1800× ( C, E ). No immunostaining was detected in sections not treated with primary antibody.

    Techniques Used: Confocal Microscopy, Derivative Assay, Mouse Assay, Injection, Labeling, Immunostaining

    21) Product Images from "Neoglycoconjugate of Tetrasaccharide Representing One Repeating Unit of the Streptococcus pneumoniae Type 14 Capsular Polysaccharide Induces the Production of Opsonizing IgG1 Antibodies and Possesses the Highest Protective Activity As Compared to Hexa- and Octasaccharide Conjugates"

    Article Title: Neoglycoconjugate of Tetrasaccharide Representing One Repeating Unit of the Streptococcus pneumoniae Type 14 Capsular Polysaccharide Induces the Production of Opsonizing IgG1 Antibodies and Possesses the Highest Protective Activity As Compared to Hexa- and Octasaccharide Conjugates

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.00659

    Inhibition of IgG1 antibodies (Abs) in mice immune sera with OS ligands and CP. Enzyme-linked immunosorbent assay inhibition assays were performed using streptavidin-coated plates with tetra-biotin, hexa-biotin, and octa-biotin adsorbed on their surfaces. (A–C) Inhibition of IgG1 Abs in the pooled sera of BALB/c mice ( n = 6) that were immunized twice intraperitoneally with glycoconjugates at 10 µg/dose. Sera were obtained 14 days after the second immunization. Serum samples for all glycoconjugates were diluted 1:4,000. The tetra-, hexa-, and octasaccharide ligands, synCP, and bacCP were used as inhibitors and applied in amounts ranging from 0 to 10 µg/well. The horizontal line indicates the IC 50 at the point of intersection of the inhibition curves. (A) Tetra-bovine serum albumin (BSA) conjugate antiserum was tested against tetra-biotin capture material. (B) Hexa-BSA conjugate antiserum was tested against hexa-biotin capture material (C) . Octa-BSA conjugate antiserum was tested against octa-biotin capture material. (D) Inhibition of IgG1 Abs was measured in the pooled sera of BALB/c mice immunized twice intraperitoneally over 2 weeks with the conjugated pneumococcal vaccine, Prevenar-13, at 1.1 µg of Streptococcus pneumoniae type 14 CP per single dose. The dilutions of sera tested against the tetra-biotin and octa-biotin coating antigens were 1:500; hexa-biotin was diluted 1:300. (E) Inhibition of IgG Abs was measured in serum harvested from rabbits that were immunized multiple times with inactivated S. pneumonia type 14 bacteria. The dilution of rabbit sera tested against tetra-biotin coating antigens was 1:300, against hexa-biotin and octa-biotin coating antigens was 1:3,000; n = 3 per data point.
    Figure Legend Snippet: Inhibition of IgG1 antibodies (Abs) in mice immune sera with OS ligands and CP. Enzyme-linked immunosorbent assay inhibition assays were performed using streptavidin-coated plates with tetra-biotin, hexa-biotin, and octa-biotin adsorbed on their surfaces. (A–C) Inhibition of IgG1 Abs in the pooled sera of BALB/c mice ( n = 6) that were immunized twice intraperitoneally with glycoconjugates at 10 µg/dose. Sera were obtained 14 days after the second immunization. Serum samples for all glycoconjugates were diluted 1:4,000. The tetra-, hexa-, and octasaccharide ligands, synCP, and bacCP were used as inhibitors and applied in amounts ranging from 0 to 10 µg/well. The horizontal line indicates the IC 50 at the point of intersection of the inhibition curves. (A) Tetra-bovine serum albumin (BSA) conjugate antiserum was tested against tetra-biotin capture material. (B) Hexa-BSA conjugate antiserum was tested against hexa-biotin capture material (C) . Octa-BSA conjugate antiserum was tested against octa-biotin capture material. (D) Inhibition of IgG1 Abs was measured in the pooled sera of BALB/c mice immunized twice intraperitoneally over 2 weeks with the conjugated pneumococcal vaccine, Prevenar-13, at 1.1 µg of Streptococcus pneumoniae type 14 CP per single dose. The dilutions of sera tested against the tetra-biotin and octa-biotin coating antigens were 1:500; hexa-biotin was diluted 1:300. (E) Inhibition of IgG Abs was measured in serum harvested from rabbits that were immunized multiple times with inactivated S. pneumonia type 14 bacteria. The dilution of rabbit sera tested against tetra-biotin coating antigens was 1:300, against hexa-biotin and octa-biotin coating antigens was 1:3,000; n = 3 per data point.

    Techniques Used: Inhibition, Mouse Assay, Enzyme-linked Immunosorbent Assay

    Anti-CP IgG1 antibody (Ab) titers in mice immunized with the glycoconjugates. BALB/c mice were immunized intraperitoneally with tetra-bovine serum albumin (BSA) (A) , hexa-BSA (B) , and octa-BSA (C) conjugates adsorbed on aluminum hydroxide twice over 14 days at 1.25–10 µg/dose (the hexa-BSA conjugate was injected at 2.5–10 µg/dose). Anti-CP IgG1 Ab titers in murine blood sera were determined by enzyme-linked immunosorbent assay 2 weeks after the second immunization. Streptococcus pneumoniae type 14 bacterial CP was used as the coating antigens. The data from two experiments were summarized. For each glycoconjugate, blood was taken from 6 to 12 mice. The data represent individual anti-CP IgG1 Ab titers, bars indicates median ± SD. Mann–Whitney Rank Sum tests were used to evaluate significance. Differences in the anti-CP IgG1 Ab titers between tetra-BSA and octa-BSA conjugates at the immunizing dose of 10 µg/mouse, * P
    Figure Legend Snippet: Anti-CP IgG1 antibody (Ab) titers in mice immunized with the glycoconjugates. BALB/c mice were immunized intraperitoneally with tetra-bovine serum albumin (BSA) (A) , hexa-BSA (B) , and octa-BSA (C) conjugates adsorbed on aluminum hydroxide twice over 14 days at 1.25–10 µg/dose (the hexa-BSA conjugate was injected at 2.5–10 µg/dose). Anti-CP IgG1 Ab titers in murine blood sera were determined by enzyme-linked immunosorbent assay 2 weeks after the second immunization. Streptococcus pneumoniae type 14 bacterial CP was used as the coating antigens. The data from two experiments were summarized. For each glycoconjugate, blood was taken from 6 to 12 mice. The data represent individual anti-CP IgG1 Ab titers, bars indicates median ± SD. Mann–Whitney Rank Sum tests were used to evaluate significance. Differences in the anti-CP IgG1 Ab titers between tetra-BSA and octa-BSA conjugates at the immunizing dose of 10 µg/mouse, * P

    Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    IgG1 antibody (Ab) titer in mice immunized with Streptococcus pneumoniae type 14 CP. BALB/c mice received two intraperitoneal immunizations with the conjugated pneumococcal vaccine Prevenar-13 with aluminum phosphate as adjuvant at 1.1 µg (content of S. pneumoniae type 14 CP) per dose. The tetra-bovine serum albumin (BSA), hexa-BSA, and octa-BSA conjugates, and synthetic (synCP) and bacterial CP (bacCP) were capture antigens, coating the enzyme-linked immunosorbent assay plates. The data are represented by individual titers of IgG1 Abs, bars indicate median ± SD. Mann–Whitney Rank Sum tests were used to determine significance. For differences in the level of Abs detected against tetra-BSA and octa-BSA conjugates, * P
    Figure Legend Snippet: IgG1 antibody (Ab) titer in mice immunized with Streptococcus pneumoniae type 14 CP. BALB/c mice received two intraperitoneal immunizations with the conjugated pneumococcal vaccine Prevenar-13 with aluminum phosphate as adjuvant at 1.1 µg (content of S. pneumoniae type 14 CP) per dose. The tetra-bovine serum albumin (BSA), hexa-BSA, and octa-BSA conjugates, and synthetic (synCP) and bacterial CP (bacCP) were capture antigens, coating the enzyme-linked immunosorbent assay plates. The data are represented by individual titers of IgG1 Abs, bars indicate median ± SD. Mann–Whitney Rank Sum tests were used to determine significance. For differences in the level of Abs detected against tetra-BSA and octa-BSA conjugates, * P

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    Anti-OS IgG1 antibody (Ab) titer in mice immunized with the OS-conjugates, CP, or bacteria. The biotinylated oligosaccharides tetra-biotin, hexa-biotin, and octa-biotin were applied as coating antigens. (A) Fourteen days after the second immunization, IgG1 Ab titers were determined in the pooled sera of BALB/c mice ( n = 6) intraperitoneally vaccinated with a single dose (10 µg) of each conjugate adsorbed on aluminum hydroxide. (B) Fourteen days after the second immunization, the IgG1 Ab titers were measured in the pooled sera of two groups of BALB/c mice ( n = 6) who were intraperitoneally vaccinated with single doses of either 1.1 or 2.2 µg (CP content) of Streptococcus pneumoniae type 14 CP-CPM 197 adsorbed on aluminum phosphate. (C) The level of IgG Abs in rabbits ( n = 2) immunized with inactive S. pneumoniae type 14 bacterial cells. Ab titers were transformed to log 10 . n = 6 assays per antiserum. The data are displayed as a mean value ± SD. Mann–Whitney Rank Sum tests were used to determine significance, * P
    Figure Legend Snippet: Anti-OS IgG1 antibody (Ab) titer in mice immunized with the OS-conjugates, CP, or bacteria. The biotinylated oligosaccharides tetra-biotin, hexa-biotin, and octa-biotin were applied as coating antigens. (A) Fourteen days after the second immunization, IgG1 Ab titers were determined in the pooled sera of BALB/c mice ( n = 6) intraperitoneally vaccinated with a single dose (10 µg) of each conjugate adsorbed on aluminum hydroxide. (B) Fourteen days after the second immunization, the IgG1 Ab titers were measured in the pooled sera of two groups of BALB/c mice ( n = 6) who were intraperitoneally vaccinated with single doses of either 1.1 or 2.2 µg (CP content) of Streptococcus pneumoniae type 14 CP-CPM 197 adsorbed on aluminum phosphate. (C) The level of IgG Abs in rabbits ( n = 2) immunized with inactive S. pneumoniae type 14 bacterial cells. Ab titers were transformed to log 10 . n = 6 assays per antiserum. The data are displayed as a mean value ± SD. Mann–Whitney Rank Sum tests were used to determine significance, * P

    Techniques Used: Mouse Assay, Transformation Assay, MANN-WHITNEY

    Inhibition of IgG antibodies (Abs) recognizing SynCP as the coating antigen with OS ligands and CP. Pooled immune sera were obtained after double intraperitoneal immunization of BALB/c mice ( n = 6 for each glycoconjugate) with the OS-bovine serum albumin (BSA) conjugates adsorbed on aluminum hydroxide (10 µg of carbohydrate/single dose). The tetra-, hexa-, and octasaccharide ligands, as well as synCP and bacCP, were used as inhibitory materials at concentrations of 0–10 µg/well. The horizontal line indicates the IC 50 level at the point of intersection of the inhibition curves. (A) Inhibition of IgG Abs was measured in tetra-BSA conjugate sera at a dilution of 1:400. (B) Inhibition of IgG Abs was measured in hexa-BSA conjugate sera at a dilution of 1:400. (C) Inhibition of IgG Abs was measured in octa-BSA conjugate sera at a dilution of 1:1,600. (D) Inhibition of IgG Abs was measured in Streptococcus pneumoniae type 14 CP-CRM 197 at a dilution of 1:200. Immune sera were obtained after immunization of mice ( n = 6) with Prevenar-13 adsorbed on aluminum phosphate at 2.2 µg (content of S. pneumoniae type 14 CP) per single dose.
    Figure Legend Snippet: Inhibition of IgG antibodies (Abs) recognizing SynCP as the coating antigen with OS ligands and CP. Pooled immune sera were obtained after double intraperitoneal immunization of BALB/c mice ( n = 6 for each glycoconjugate) with the OS-bovine serum albumin (BSA) conjugates adsorbed on aluminum hydroxide (10 µg of carbohydrate/single dose). The tetra-, hexa-, and octasaccharide ligands, as well as synCP and bacCP, were used as inhibitory materials at concentrations of 0–10 µg/well. The horizontal line indicates the IC 50 level at the point of intersection of the inhibition curves. (A) Inhibition of IgG Abs was measured in tetra-BSA conjugate sera at a dilution of 1:400. (B) Inhibition of IgG Abs was measured in hexa-BSA conjugate sera at a dilution of 1:400. (C) Inhibition of IgG Abs was measured in octa-BSA conjugate sera at a dilution of 1:1,600. (D) Inhibition of IgG Abs was measured in Streptococcus pneumoniae type 14 CP-CRM 197 at a dilution of 1:200. Immune sera were obtained after immunization of mice ( n = 6) with Prevenar-13 adsorbed on aluminum phosphate at 2.2 µg (content of S. pneumoniae type 14 CP) per single dose.

    Techniques Used: Inhibition, Mouse Assay

    22) Product Images from "Quantitative Analysis of Cellular Proteome Alterations in CDV-Infected Mink Lung Epithelial Cells"

    Article Title: Quantitative Analysis of Cellular Proteome Alterations in CDV-Infected Mink Lung Epithelial Cells

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2017.02564

    CDV infection induces the phosphorylation and nuclear translocation of NF-κB P65 and the degradation of IκB-α proteins (A) CDV infection strengthened NF-κB P65 phosphorylation and IκB-α degradation. Mv.1.Lu cells were infected with 2 MOI PS or CDV 3 . At 24 hpi, cells were gathered for detecting the expression levels of phosphorylated NF-κB P65 protein and IκB-α protein. (B) CDV infection facilitated NF-κB P65 nuclear translocation. Mv.1.Lu cells were infected with 2 MOI PS, CDV 3 or mock-infected. At 24 hpi, the cells were fixed and incubated with rabbit polyclonal antibody specific to mink NF-κB P65 and mouse monoclonal antibody specific to CDV N protein, then incubated with FITC-labeled goat anti rabbit IgG and Cy3-labeled goat anti mouse IgG, respectively. Cell nuclei were stained by DAPI. The fluorescent images were analyzed under a confocal microscopy (Leica, Germany).
    Figure Legend Snippet: CDV infection induces the phosphorylation and nuclear translocation of NF-κB P65 and the degradation of IκB-α proteins (A) CDV infection strengthened NF-κB P65 phosphorylation and IκB-α degradation. Mv.1.Lu cells were infected with 2 MOI PS or CDV 3 . At 24 hpi, cells were gathered for detecting the expression levels of phosphorylated NF-κB P65 protein and IκB-α protein. (B) CDV infection facilitated NF-κB P65 nuclear translocation. Mv.1.Lu cells were infected with 2 MOI PS, CDV 3 or mock-infected. At 24 hpi, the cells were fixed and incubated with rabbit polyclonal antibody specific to mink NF-κB P65 and mouse monoclonal antibody specific to CDV N protein, then incubated with FITC-labeled goat anti rabbit IgG and Cy3-labeled goat anti mouse IgG, respectively. Cell nuclei were stained by DAPI. The fluorescent images were analyzed under a confocal microscopy (Leica, Germany).

    Techniques Used: Infection, Translocation Assay, Expressing, Incubation, Labeling, Staining, Confocal Microscopy

    23) Product Images from "Pathogenic Lifestyles of E. coli Pathotypes in a Standardized Epithelial Cell Model Influence Inflammatory Signaling Pathways and Cytokines Secretion"

    Article Title: Pathogenic Lifestyles of E. coli Pathotypes in a Standardized Epithelial Cell Model Influence Inflammatory Signaling Pathways and Cytokines Secretion

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2016.00120

    Reproduction of the phenotypic features induced by different E. coli pathotypes and nuclear translocation of ERK1/2 in HT-29 cells . HT-29 cells were infected (MOI 10) during 4 h with the different E. coli pathotypes as indicated. As controls, cells were treated with E. coli HB101, a non-pathogenic strain, or with EGF, an ERK1/2 translocation inducer. After treatment, cells were processed for confocal microscopy as indicated in Figure 1 , but ERK1/2 translocation was detected by immunofluorescence using anti-phospho-ERK1/2 antibodies followed by FITC-goat anti-mouse IgG antibodies. Arrowheads indicate the location of bacterial adhesion and arrows point out cytoskeleton rearrangements (pedestal formation induced by EPEC, EHEC), rounding cell (EAEC), and cell invasion (intracellular actin tails by EIEC and S. flexneri ). Bar 15 μm.
    Figure Legend Snippet: Reproduction of the phenotypic features induced by different E. coli pathotypes and nuclear translocation of ERK1/2 in HT-29 cells . HT-29 cells were infected (MOI 10) during 4 h with the different E. coli pathotypes as indicated. As controls, cells were treated with E. coli HB101, a non-pathogenic strain, or with EGF, an ERK1/2 translocation inducer. After treatment, cells were processed for confocal microscopy as indicated in Figure 1 , but ERK1/2 translocation was detected by immunofluorescence using anti-phospho-ERK1/2 antibodies followed by FITC-goat anti-mouse IgG antibodies. Arrowheads indicate the location of bacterial adhesion and arrows point out cytoskeleton rearrangements (pedestal formation induced by EPEC, EHEC), rounding cell (EAEC), and cell invasion (intracellular actin tails by EIEC and S. flexneri ). Bar 15 μm.

    Techniques Used: Translocation Assay, Infection, Confocal Microscopy, Immunofluorescence

    Reproduction of the phenotypic features (adhesion, cytotoxicity and actin cytoskeleton rearrangements) induced by different E. coli pathotypes and nuclear translocation of NF-κB in HT-29 cells as intestinal model . HT-29 cells were infected (MOI 10) during 4 h with the different E. coli pathotypes as indicated. As controls, cells were treated with E. coli HB101, a non-pathogenic strain, or with TNF-α, a NF-κB translocation inducer. After treatment, cells were washed, fixed and stained with rhodamine-phalloidin for actin filaments and TO-PRO-3 for DNA detection, while NF-κB translocation was detected by immunofluorescence with anti-phospho-NF-κB (phospho-p65) antibodies followed by FITC-goat anti-rabbit IgG antibodies. The preparations were analyzed and documented with a confocal microscope (63X). Arrowheads indicate the location of bacterial adhesion and arrows point out cytoskeleton rearrangements (pedestal formation induced by EPEC, EHEC), rounding cell (EAEC), and cell invasion (intracellular actin tails by EIEC and S. flexneri ). Bar 15 μm.
    Figure Legend Snippet: Reproduction of the phenotypic features (adhesion, cytotoxicity and actin cytoskeleton rearrangements) induced by different E. coli pathotypes and nuclear translocation of NF-κB in HT-29 cells as intestinal model . HT-29 cells were infected (MOI 10) during 4 h with the different E. coli pathotypes as indicated. As controls, cells were treated with E. coli HB101, a non-pathogenic strain, or with TNF-α, a NF-κB translocation inducer. After treatment, cells were washed, fixed and stained with rhodamine-phalloidin for actin filaments and TO-PRO-3 for DNA detection, while NF-κB translocation was detected by immunofluorescence with anti-phospho-NF-κB (phospho-p65) antibodies followed by FITC-goat anti-rabbit IgG antibodies. The preparations were analyzed and documented with a confocal microscope (63X). Arrowheads indicate the location of bacterial adhesion and arrows point out cytoskeleton rearrangements (pedestal formation induced by EPEC, EHEC), rounding cell (EAEC), and cell invasion (intracellular actin tails by EIEC and S. flexneri ). Bar 15 μm.

    Techniques Used: Translocation Assay, Infection, Staining, Immunofluorescence, Microscopy

    24) Product Images from "Enhanced viral-mediated cochlear gene delivery in adult mice by combining canal fenestration with round window membrane inoculation"

    Article Title: Enhanced viral-mediated cochlear gene delivery in adult mice by combining canal fenestration with round window membrane inoculation

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-21233-z

    Vestibular organs are transduced following the RWM + CF injection. ( a – c ) Representative images of whole mounts of the CA of the PSCC, saccule, utricle and CAs of the LSCC and ASCC as indicated. Tissue was harvested 2 weeks after the RWM + CF injection (at P15–16), stained with Alexa Fluor 568-phalloidin (red) for labelling filamentous actin, and imaged for native eGFP (green). High magnification views of the regions marked with white dotted squares in the PSCC are shown separately and stained with Myo7a (gray) for labelling hair cells and imaged for native eGFP (green).
    Figure Legend Snippet: Vestibular organs are transduced following the RWM + CF injection. ( a – c ) Representative images of whole mounts of the CA of the PSCC, saccule, utricle and CAs of the LSCC and ASCC as indicated. Tissue was harvested 2 weeks after the RWM + CF injection (at P15–16), stained with Alexa Fluor 568-phalloidin (red) for labelling filamentous actin, and imaged for native eGFP (green). High magnification views of the regions marked with white dotted squares in the PSCC are shown separately and stained with Myo7a (gray) for labelling hair cells and imaged for native eGFP (green).

    Techniques Used: Injection, Staining

    25) Product Images from "Tubulin detyrosination promotes human trophoblast syncytium formation"

    Article Title: Tubulin detyrosination promotes human trophoblast syncytium formation

    Journal: Journal of Molecular Cell Biology

    doi: 10.1093/jmcb/mjz084

    Membrane proteins required for fusion are enriched in detyr-α-tub-overexpressing cell membranes. ( A ) Time-lapse images of BeWo cells carrying EGFP-α-tubulin or mCherry-α-tubulin. The cells were treated with forskolin to induce fusion. The white arrowheads indicate where fusion occurred. Scale bar, 10 μm. ( B ) SR-SIM. Alexa Fluor 488 (green) and Alexa Fluor 555 (red) were used to label α-tubulin and detyr-α-tub, respectively, in cells after a 48-h forskolin treatment. Scale bar, 2.5 μm. ( C ) The influences of TTL overexpression or knockdown on the expression of cell surface proteins in different cell fractions of BeWo cells treated with forskolin. The cell surface proteins were isolated and examined with the indicated antibodies. Na + /K + -ATPase served as a plasma membrane loading control. Whole-cell lysates were harvested and examined with the indicated antibodies. GAPDH served as a loading control.
    Figure Legend Snippet: Membrane proteins required for fusion are enriched in detyr-α-tub-overexpressing cell membranes. ( A ) Time-lapse images of BeWo cells carrying EGFP-α-tubulin or mCherry-α-tubulin. The cells were treated with forskolin to induce fusion. The white arrowheads indicate where fusion occurred. Scale bar, 10 μm. ( B ) SR-SIM. Alexa Fluor 488 (green) and Alexa Fluor 555 (red) were used to label α-tubulin and detyr-α-tub, respectively, in cells after a 48-h forskolin treatment. Scale bar, 2.5 μm. ( C ) The influences of TTL overexpression or knockdown on the expression of cell surface proteins in different cell fractions of BeWo cells treated with forskolin. The cell surface proteins were isolated and examined with the indicated antibodies. Na + /K + -ATPase served as a plasma membrane loading control. Whole-cell lysates were harvested and examined with the indicated antibodies. GAPDH served as a loading control.

    Techniques Used: Over Expression, Expressing, Isolation

    Augmented detyrosination of α-tubulin during spontaneous fusion of primary CTBs. ( A ) Illustrative model depicting BeWo cell fusion. ( B ) Upper panel: schematic illustration of spontaneous syncytialization of human primary CTBs after isolation. Lower panel: human trophoblasts stained at 0 and 72 h of culture for E-cadherin and nuclei. Scale bar, 50 μm. ( C ) Results of western blotting using the indicated antibodies on primary CTBs from the indicated gestational ages and their corresponding syncytia after 72 h of incubation. GAPDH was used as a loading control (here and hereafter). The colored lines represent groups of corresponding gestational ages. ( D ) Quantification of the western blot data in C . The band intensities detected with anti-α-tubulin and anti-detyr-α-tub antibodies were normalized to that of GAPDH as a control. The dots and squares representing individual samples from patients are colored according to the matching samples at different time points. The horizontal lines represent the means. ( E ) For confocal microscopy, Alexa Fluor 488 (green) was used to label TTL. Alexa Fluor 555 (red) was used to label Syncytin-2, active caspase-8, or CK7. Scale bar, 10 μm. The arrows indicate a layer of mononucleated CTBs and a layer of multinucleated STB.
    Figure Legend Snippet: Augmented detyrosination of α-tubulin during spontaneous fusion of primary CTBs. ( A ) Illustrative model depicting BeWo cell fusion. ( B ) Upper panel: schematic illustration of spontaneous syncytialization of human primary CTBs after isolation. Lower panel: human trophoblasts stained at 0 and 72 h of culture for E-cadherin and nuclei. Scale bar, 50 μm. ( C ) Results of western blotting using the indicated antibodies on primary CTBs from the indicated gestational ages and their corresponding syncytia after 72 h of incubation. GAPDH was used as a loading control (here and hereafter). The colored lines represent groups of corresponding gestational ages. ( D ) Quantification of the western blot data in C . The band intensities detected with anti-α-tubulin and anti-detyr-α-tub antibodies were normalized to that of GAPDH as a control. The dots and squares representing individual samples from patients are colored according to the matching samples at different time points. The horizontal lines represent the means. ( E ) For confocal microscopy, Alexa Fluor 488 (green) was used to label TTL. Alexa Fluor 555 (red) was used to label Syncytin-2, active caspase-8, or CK7. Scale bar, 10 μm. The arrows indicate a layer of mononucleated CTBs and a layer of multinucleated STB.

    Techniques Used: Isolation, Staining, Western Blot, Incubation, Confocal Microscopy

    26) Product Images from "Three YXXL Sequences of a Bovine Leukemia Virus Transmembrane Protein are Independently Required for Fusion Activity by Controlling Expression on the Cell Membrane"

    Article Title: Three YXXL Sequences of a Bovine Leukemia Virus Transmembrane Protein are Independently Required for Fusion Activity by Controlling Expression on the Cell Membrane

    Journal: Viruses

    doi: 10.3390/v11121140

    Effect on the localization of gp51 by mutant forms of the Env expression plasmid pEnv. HeLa cells (1.0 × 10 5 ) were seeded on a coverslip in a 12 well plate the day prior to transfection and transfected with 2 µg of either wild-type pEnv, mutant pEnv, or the control pME-18neo using 8 μL of FuGENE HD. ( A ) To detect cell surface gp51, cells were fixed and stained using an anti-gp51 MAb, followed by Alexa Fluor 488-conjugated anti-mouse IgG. ( B ) To detect intracellular gp51, cells were fixed, permeabilized with 0.5% Triton X-100, and stained with an anti-gp51 MAb followed by Alexa Fluor 488-conjugated anti-mouse IgG. ( A , B ) Fluorescence intensity maps were plotted for linear transects drawn through the nuclei by line scan measurements through each cell using FV10-ASW 4.02 microscope software, and fluorescence intensities on the cell surface were measured. The width of each line was thinner than 1 pixel. Peak membrane intensity was normalized by the mean intensity of pEnv for each experiment. The results show the relative intensities of at least 50 cells expressing gp51 over six independent experiments. Each column and error bar represents the mean ± SD of intensity for all cells. All values were analyzed by two-way ANOVA with Dunnett’s test. The asterisk indicates a statistically significant difference (* p
    Figure Legend Snippet: Effect on the localization of gp51 by mutant forms of the Env expression plasmid pEnv. HeLa cells (1.0 × 10 5 ) were seeded on a coverslip in a 12 well plate the day prior to transfection and transfected with 2 µg of either wild-type pEnv, mutant pEnv, or the control pME-18neo using 8 μL of FuGENE HD. ( A ) To detect cell surface gp51, cells were fixed and stained using an anti-gp51 MAb, followed by Alexa Fluor 488-conjugated anti-mouse IgG. ( B ) To detect intracellular gp51, cells were fixed, permeabilized with 0.5% Triton X-100, and stained with an anti-gp51 MAb followed by Alexa Fluor 488-conjugated anti-mouse IgG. ( A , B ) Fluorescence intensity maps were plotted for linear transects drawn through the nuclei by line scan measurements through each cell using FV10-ASW 4.02 microscope software, and fluorescence intensities on the cell surface were measured. The width of each line was thinner than 1 pixel. Peak membrane intensity was normalized by the mean intensity of pEnv for each experiment. The results show the relative intensities of at least 50 cells expressing gp51 over six independent experiments. Each column and error bar represents the mean ± SD of intensity for all cells. All values were analyzed by two-way ANOVA with Dunnett’s test. The asterisk indicates a statistically significant difference (* p

    Techniques Used: Mutagenesis, Expressing, Plasmid Preparation, Transfection, Staining, Fluorescence, Microscopy, Software

    Effect on the localization of gp51 by mutant forms of the infectious molecular clone pBLV-IF2. HeLa cells (1.0 × 10 5 ) were seeded on a coverslip in a 12 well plate the day prior to transfection and transfected with 2 µg of either wild-type pBLV-IF2, mutant pBLV-IF2, or control pBluescript II SK (−) using 8 μL of FuGENE HD. The transfection efficiency was similar among all mutant pBLV-IF2s as evaluated according to the ratio of GFP-expressing HeLa cells determined via FACSCalibur™ flow cytometry. ( A ) To detect cell surface gp51, cells were fixed and stained with anti-gp51 MAb, followed by Alexa Fluor 488-conjugated anti-Mouse IgG, then stained with Hoechst 33342 and observed using an FV-1000 fluorescence microscope (right panel). ( B ) To detect intracellular Env protein, cells were fixed, permeabilized with 0.5% Triton X-100, stained with anti-BLVgp51 MAb followed by Alexa Fluor 488-conjugated anti-Mouse IgG, and observed using an FV-1000 fluorescence microscope (right panel). ( A , B ) Fluorescence intensity maps were plotted for linear transects drawn through the nuclei by line scan measurements through each cell using FV10-ASW 4.02 microscope software, and fluorescence intensities on the cell surface were measured. The width of each line was thinner than 1 pixel. Peak membrane intensity was normalized by the mean intensity of pBLV-IF2 for each experiment. The results show the relative intensities of at least 50 cells expressing gp51 over seven independent experiments. Each column and error bar represents the mean ± SD of intensity for all cells. All values were analyzed by two-way ANOVA with Dunnett’s test. The asterisk indicates a statistically significant difference (* p
    Figure Legend Snippet: Effect on the localization of gp51 by mutant forms of the infectious molecular clone pBLV-IF2. HeLa cells (1.0 × 10 5 ) were seeded on a coverslip in a 12 well plate the day prior to transfection and transfected with 2 µg of either wild-type pBLV-IF2, mutant pBLV-IF2, or control pBluescript II SK (−) using 8 μL of FuGENE HD. The transfection efficiency was similar among all mutant pBLV-IF2s as evaluated according to the ratio of GFP-expressing HeLa cells determined via FACSCalibur™ flow cytometry. ( A ) To detect cell surface gp51, cells were fixed and stained with anti-gp51 MAb, followed by Alexa Fluor 488-conjugated anti-Mouse IgG, then stained with Hoechst 33342 and observed using an FV-1000 fluorescence microscope (right panel). ( B ) To detect intracellular Env protein, cells were fixed, permeabilized with 0.5% Triton X-100, stained with anti-BLVgp51 MAb followed by Alexa Fluor 488-conjugated anti-Mouse IgG, and observed using an FV-1000 fluorescence microscope (right panel). ( A , B ) Fluorescence intensity maps were plotted for linear transects drawn through the nuclei by line scan measurements through each cell using FV10-ASW 4.02 microscope software, and fluorescence intensities on the cell surface were measured. The width of each line was thinner than 1 pixel. Peak membrane intensity was normalized by the mean intensity of pBLV-IF2 for each experiment. The results show the relative intensities of at least 50 cells expressing gp51 over seven independent experiments. Each column and error bar represents the mean ± SD of intensity for all cells. All values were analyzed by two-way ANOVA with Dunnett’s test. The asterisk indicates a statistically significant difference (* p

    Techniques Used: Mutagenesis, Transfection, Expressing, Flow Cytometry, Cytometry, Staining, Fluorescence, Microscopy, Software

    Localization of gp51 in the early endosome and trans-Golgi network. HeLa cells (1.0 × 10 5 ) were seeded on a coverslip in a 12 well plate the day prior to transfection and transfected with 2 µg of either wild-type pBLV-IF2, mutant pBLV-IF2, or control pBluescript II SK (−) using 8 μL of FuGENE HD. ( A ) To detect the localization of gp51 in the early endosome, cells were fixed, permeabilized with 0.5% Triton X-100, and stained with anti-BLV gp51 MAb followed by Alexa Fluor 594-conjugated anti-mouse IgG and anti-EEA1 polyclonal antibody (PAb), followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (upper panel). The colocalization index between gp51 and EEA1 was calculated using Villalta’s algorithm with FV10-ASW 4.02 microscope software (lower panel). The results show the relative colocalization index between gp51 and EEA1 of at least 50 cells over seven independent experiments. Each column and error bar represents the mean ± SD of intensity for all cells. All values were analyzed by two-way ANOVA with Dunnett’s test. ( B ) To detect the localization of gp51 in the trans-Golgi network, cells were fixed, permeabilized with 0.5% Triton X-100, and stained with anti-BLV gp51 MAb followed by Alexa Fluor 594-conjugated goat anti-mouse, and anti-TGN46 PAb followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (upper panel). The colocalization index between gp51 and TGN46 was calculated using Villalta’s algorithm on FV10-ASW 4.02 microscope software (Lower panel). The results show the relative colocalization index between gp51 and TGN46 of at least 50 cells over three independent experiments. Each column and error bar represents the mean ± SD of intensity for all cells. All values were analyzed by two-way ANOVA with Dunnett’s test. The asterisk indicates a statistically significant difference (* p
    Figure Legend Snippet: Localization of gp51 in the early endosome and trans-Golgi network. HeLa cells (1.0 × 10 5 ) were seeded on a coverslip in a 12 well plate the day prior to transfection and transfected with 2 µg of either wild-type pBLV-IF2, mutant pBLV-IF2, or control pBluescript II SK (−) using 8 μL of FuGENE HD. ( A ) To detect the localization of gp51 in the early endosome, cells were fixed, permeabilized with 0.5% Triton X-100, and stained with anti-BLV gp51 MAb followed by Alexa Fluor 594-conjugated anti-mouse IgG and anti-EEA1 polyclonal antibody (PAb), followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (upper panel). The colocalization index between gp51 and EEA1 was calculated using Villalta’s algorithm with FV10-ASW 4.02 microscope software (lower panel). The results show the relative colocalization index between gp51 and EEA1 of at least 50 cells over seven independent experiments. Each column and error bar represents the mean ± SD of intensity for all cells. All values were analyzed by two-way ANOVA with Dunnett’s test. ( B ) To detect the localization of gp51 in the trans-Golgi network, cells were fixed, permeabilized with 0.5% Triton X-100, and stained with anti-BLV gp51 MAb followed by Alexa Fluor 594-conjugated goat anti-mouse, and anti-TGN46 PAb followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (upper panel). The colocalization index between gp51 and TGN46 was calculated using Villalta’s algorithm on FV10-ASW 4.02 microscope software (Lower panel). The results show the relative colocalization index between gp51 and TGN46 of at least 50 cells over three independent experiments. Each column and error bar represents the mean ± SD of intensity for all cells. All values were analyzed by two-way ANOVA with Dunnett’s test. The asterisk indicates a statistically significant difference (* p

    Techniques Used: Transfection, Mutagenesis, Staining, Microscopy, Software

    27) Product Images from "Impact of angiotensin-converting enzyme inhibition on platelet tissue factor expression in stroke-prone rats"

    Article Title: Impact of angiotensin-converting enzyme inhibition on platelet tissue factor expression in stroke-prone rats

    Journal: Journal of Hypertension

    doi: 10.1097/HJH.0000000000001702

    Flow cytometry analysis of cell-surface tissue factor expression in rat and human platelets stimulated with angiotensin II. Platelet-rich plasma from normotensive Wistar Kyoto rats was stimulated with increasing concentration of collagen (a) or, a selective AT1 agonist (b), and stained with polyclonal antitissue factor antibody labeled with Alexa Fluor 633. The effect of AngII on human platelets is also reported (c). Mean percentage of tissue factor-positive cells ± SD, in each experimental setting, is reported as histogram. ANOVA was applied for statistical analysis. P values were obtained by post-hoc test.
    Figure Legend Snippet: Flow cytometry analysis of cell-surface tissue factor expression in rat and human platelets stimulated with angiotensin II. Platelet-rich plasma from normotensive Wistar Kyoto rats was stimulated with increasing concentration of collagen (a) or, a selective AT1 agonist (b), and stained with polyclonal antitissue factor antibody labeled with Alexa Fluor 633. The effect of AngII on human platelets is also reported (c). Mean percentage of tissue factor-positive cells ± SD, in each experimental setting, is reported as histogram. ANOVA was applied for statistical analysis. P values were obtained by post-hoc test.

    Techniques Used: Flow Cytometry, Cytometry, Expressing, Concentration Assay, Staining, Labeling

    28) Product Images from "A Stem-Cell-Derived Platform Enables Complete Cryptosporidium Development In Vitro and Genetic Tractability"

    Article Title: A Stem-Cell-Derived Platform Enables Complete Cryptosporidium Development In Vitro and Genetic Tractability

    Journal: Cell Host & Microbe

    doi: 10.1016/j.chom.2019.05.007

    In vitro production of oocysts in ALI culture (A) Effect of filtration on removal of residual C. parvum oocysts. Oocysts were excysted, filtered using the indicated pore sizes, added to PLL-coated coverslips, and stained with Crypt-a-glo directly conjugated to FITC. The number of residual oocysts found in filtered samples were counted from replicate 63× fields from a representative experiment. (B) Detection of C. parvum oocysts in ALI cultures. Transwells were infected on day 3 post top medium removal with 1 μm-filtered sporozoites. On specified days post infection, transwells were fixed and stained with Crypt-a-glo directly conjugated to FITC and Pan Cp, detected with goat anti-rabbit IgG Alexa Fluor 568. Oocyst numbers per 63× field were counted for three independent experiments (Exp A, Exp B, Exp C). The combined mean of each time point was compared to the combined mean of day 1 using a two-way ANOVA corrected for multiple comparisons using the Dunnett method, ∗∗∗∗ p
    Figure Legend Snippet: In vitro production of oocysts in ALI culture (A) Effect of filtration on removal of residual C. parvum oocysts. Oocysts were excysted, filtered using the indicated pore sizes, added to PLL-coated coverslips, and stained with Crypt-a-glo directly conjugated to FITC. The number of residual oocysts found in filtered samples were counted from replicate 63× fields from a representative experiment. (B) Detection of C. parvum oocysts in ALI cultures. Transwells were infected on day 3 post top medium removal with 1 μm-filtered sporozoites. On specified days post infection, transwells were fixed and stained with Crypt-a-glo directly conjugated to FITC and Pan Cp, detected with goat anti-rabbit IgG Alexa Fluor 568. Oocyst numbers per 63× field were counted for three independent experiments (Exp A, Exp B, Exp C). The combined mean of each time point was compared to the combined mean of day 1 using a two-way ANOVA corrected for multiple comparisons using the Dunnett method, ∗∗∗∗ p

    Techniques Used: In Vitro, Filtration, Staining, Infection

    ALI Transwells support genetic crosses of C. parvum in vitro (A) Diagram of targeting construct designed to replace the endogenous tk locus (cgd5_4440) with GFP and Nluc-P2A-Neo R cassette. (B) Relative luminescence normalized to total number of parasites in ALI transwells at 1 and 4 days post infection (dpi). Transwells were infected with ∼1 × 10 7 unfiltered sporozoites that were electroporated with the TK-GFP-Nluc-P2A-neo-TK reporter and a Cas9 plasmid with a TK gRNA. Transwells were cultured in medium containing PBS (light green) as a control or 20 mM paromomycin (PRM, dark green). Data plotted as mean ± S.D. from two transwells per time point from a representative experiment. Nonsignificant (p = 0.11), unpaired Student’s t test between PBS and PRM-treated transwells 4 dpi. (C) Image of whole-mount ALI transwells 5 dpi with transfected C. parvum from same experiment as (B) stained with anti-GFP followed by goat anti-rabbit IgG Alexa Fluor 488. Scale bar, 10 μm. (D) Merged image of (C) with a Pan Cp polyclonal antibody, which recognizes all C. parvum stages, followed by goat antirat IgG Alexa Fluor 568. Scale bar, 10 μm. (E) Diagram of targeting construct designed to replace the endogenous uprt locus (cgd1_1900) with mCherry and Nluc-P2A-Neo R cassette as (A). (F) Relative luminescence normalized to total number of parasites in ALI transwells at 1 and 3 dpi. Transwells were infected with ∼1 × 10 7 unfiltered sporozoites per transwell that were electroporated with the UPRT-mCh-Nluc-P2A-neo-UPRT reporter and a Cas9 plasmid with a UPRT gRNA. Transwells were cultured in medium containing PBS (pink) as a control or 20 mM PRM (red). Data plotted as mean ± S.D. from two transwells per time point from a representative experiment. ∗ p
    Figure Legend Snippet: ALI Transwells support genetic crosses of C. parvum in vitro (A) Diagram of targeting construct designed to replace the endogenous tk locus (cgd5_4440) with GFP and Nluc-P2A-Neo R cassette. (B) Relative luminescence normalized to total number of parasites in ALI transwells at 1 and 4 days post infection (dpi). Transwells were infected with ∼1 × 10 7 unfiltered sporozoites that were electroporated with the TK-GFP-Nluc-P2A-neo-TK reporter and a Cas9 plasmid with a TK gRNA. Transwells were cultured in medium containing PBS (light green) as a control or 20 mM paromomycin (PRM, dark green). Data plotted as mean ± S.D. from two transwells per time point from a representative experiment. Nonsignificant (p = 0.11), unpaired Student’s t test between PBS and PRM-treated transwells 4 dpi. (C) Image of whole-mount ALI transwells 5 dpi with transfected C. parvum from same experiment as (B) stained with anti-GFP followed by goat anti-rabbit IgG Alexa Fluor 488. Scale bar, 10 μm. (D) Merged image of (C) with a Pan Cp polyclonal antibody, which recognizes all C. parvum stages, followed by goat antirat IgG Alexa Fluor 568. Scale bar, 10 μm. (E) Diagram of targeting construct designed to replace the endogenous uprt locus (cgd1_1900) with mCherry and Nluc-P2A-Neo R cassette as (A). (F) Relative luminescence normalized to total number of parasites in ALI transwells at 1 and 3 dpi. Transwells were infected with ∼1 × 10 7 unfiltered sporozoites per transwell that were electroporated with the UPRT-mCh-Nluc-P2A-neo-UPRT reporter and a Cas9 plasmid with a UPRT gRNA. Transwells were cultured in medium containing PBS (pink) as a control or 20 mM PRM (red). Data plotted as mean ± S.D. from two transwells per time point from a representative experiment. ∗ p

    Techniques Used: In Vitro, Construct, Infection, Plasmid Preparation, Cell Culture, Transfection, Staining

    Infectivity of oocysts produced in ALI cultures (A) Detection of oocysts in ALI cultures treated with bleach. ALI transwell cultures were infected with 1 μm-filtered sporozoites. On days 1 and 3 postinfection, transwells were bleached, washed, pelleted, adhered to PLL-coated coverslips, and stained with Pan Cp followed by goat anti-rabbit Alexa Fluor 568 and Crypt-a-glo directly conjugated to FITC. Each data point is the number of oocysts in a single 63X field from a single representative experiment. Data were analyzed using a Mann-Whitney U test. ∗∗∗∗ for replicate experiment. (B) Images of bleached oocysts isolated from ALI cultures at day 3 postinfection as described in (A). Oocysts were stained with Pan Cp followed by goat anti-rabbit IgG Alexa Fluor 568, Crypt-a-glo directly conjugated to FITC, and Hoechst. Scale bars, 5 μm. (C and D) Infectivity of bleached ALI monolayers for mice. ALI transwell cultures were infected with 1 μm-filtered sporozoites. On days 1 and 3 postinfection, transwells were bleached, washed, and the material was orally gavaged into naïve Ifngr1 −/− mice. (C) Survival curves of the mice analyzed using the log-rank (Mantel-Cox) test, ∗∗ p
    Figure Legend Snippet: Infectivity of oocysts produced in ALI cultures (A) Detection of oocysts in ALI cultures treated with bleach. ALI transwell cultures were infected with 1 μm-filtered sporozoites. On days 1 and 3 postinfection, transwells were bleached, washed, pelleted, adhered to PLL-coated coverslips, and stained with Pan Cp followed by goat anti-rabbit Alexa Fluor 568 and Crypt-a-glo directly conjugated to FITC. Each data point is the number of oocysts in a single 63X field from a single representative experiment. Data were analyzed using a Mann-Whitney U test. ∗∗∗∗ for replicate experiment. (B) Images of bleached oocysts isolated from ALI cultures at day 3 postinfection as described in (A). Oocysts were stained with Pan Cp followed by goat anti-rabbit IgG Alexa Fluor 568, Crypt-a-glo directly conjugated to FITC, and Hoechst. Scale bars, 5 μm. (C and D) Infectivity of bleached ALI monolayers for mice. ALI transwell cultures were infected with 1 μm-filtered sporozoites. On days 1 and 3 postinfection, transwells were bleached, washed, and the material was orally gavaged into naïve Ifngr1 −/− mice. (C) Survival curves of the mice analyzed using the log-rank (Mantel-Cox) test, ∗∗ p

    Techniques Used: Infection, Produced, Staining, MANN-WHITNEY, Isolation, Mouse Assay

    29) Product Images from "The extradomain a of fibronectin enhances the efficacy of lipopolysaccharide defective Salmonella bacterins as vaccines in mice"

    Article Title: The extradomain a of fibronectin enhances the efficacy of lipopolysaccharide defective Salmonella bacterins as vaccines in mice

    Journal: Veterinary Research

    doi: 10.1186/1297-9716-43-31

    EDAvidin binding to biotinylated bacterins in ELISA. ELISA plates coated with biotinylated (Biot) or not biotinylated control (B-SEΔ waaL , B-SEΔ gal and B-SEwt) bacterins were incubated with EDAvidin or EDA alone (control). Binding was monitored using a rabbit anti-EDA polyclonal antibody and an anti-rabbit whole IgG horseradish-peroxidase-conjugated second antibody. The O.D. values at 405 nm (mean ± SD) are represented.
    Figure Legend Snippet: EDAvidin binding to biotinylated bacterins in ELISA. ELISA plates coated with biotinylated (Biot) or not biotinylated control (B-SEΔ waaL , B-SEΔ gal and B-SEwt) bacterins were incubated with EDAvidin or EDA alone (control). Binding was monitored using a rabbit anti-EDA polyclonal antibody and an anti-rabbit whole IgG horseradish-peroxidase-conjugated second antibody. The O.D. values at 405 nm (mean ± SD) are represented.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation

    30) Product Images from "Molecular characterization and Functional Analysis of the PilQ380-706: a Novel Secretin Domain in Pseudomonas aeruginosa"

    Article Title: Molecular characterization and Functional Analysis of the PilQ380-706: a Novel Secretin Domain in Pseudomonas aeruginosa

    Journal: Avicenna Journal of Medical Biotechnology

    doi:

    Western blot analysis of the expressed r-PilQ 380-706 protein in E. coli BL21. After running the SDS-PAGE, the protein transferred onto PVDF membrane and detected with HRP-conjugated goat anti-rabbit IgG. (lane 1) total cell lysate of non-induced bacteria; (lane 2) total cell lysate of bacteria after 4 hr induction; (lane 3 and 4) purified r-PilQ 380-706 by Ni 2+ -affinity chromatography.
    Figure Legend Snippet: Western blot analysis of the expressed r-PilQ 380-706 protein in E. coli BL21. After running the SDS-PAGE, the protein transferred onto PVDF membrane and detected with HRP-conjugated goat anti-rabbit IgG. (lane 1) total cell lysate of non-induced bacteria; (lane 2) total cell lysate of bacteria after 4 hr induction; (lane 3 and 4) purified r-PilQ 380-706 by Ni 2+ -affinity chromatography.

    Techniques Used: Western Blot, SDS Page, Purification, Affinity Chromatography

    31) Product Images from "Human Papillomavirus Major Capsid Protein L1 Remains Associated with the Incoming Viral Genome throughout the Entry Process"

    Article Title: Human Papillomavirus Major Capsid Protein L1 Remains Associated with the Incoming Viral Genome throughout the Entry Process

    Journal: Journal of Virology

    doi: 10.1128/JVI.00537-17

    Some full-length L1 protein accompanies the viral genome to the nucleus. (A) HeLa cells were infected with HPV16 pseudovirus with or without 1.5 μM Eg5 inhibitor III (Eg5i) and tracked via live-cell imaging using the IncuCyte Zoom for 48 h. Note that the images are depicted at 18 hpi. (B) HaCaT cells were infected with EdU-labeled HPV16 pseudovirus in the presence of 1.5 μM Eg5i. The cells were fixed at 24 hpi and permeabilized with either digitonin at 0.625 μg/ml or 0.5% TX-100 and then treated with AF555 (green) in Click-iT reaction buffer. The cells were permeabilized again with 0.5% TX-100 and treated with AF647 (red) in Click-iT reaction buffer. Lastly, the cells were mounted in DAPI (blue). (C) HaCaT cells were infected with EdU-labeled pseudovirus and treated with 1.5 μM Eg5i. At 24 hpi, the cells were fixed and permeabilized in 0.5% TX-100. Next, the cells were treated with AF555 (red) in Click-iT reaction buffer, followed by incubation with AF488-conjugated anti-α-tubulin (white) and MAb 33L1-7 (green). Lastly, the cells were mounted in DAPI (blue). Note the colocalization between EdU and L1 signal denoted by white arrows. (D) HeLa cells were infected with HPV16 pseudovirus in the presence of 1.5 μM Eg5i. Cells were trypsinized for 2 min, and monoastral cells were collected. Next, the cells were treated with 15 μl of 0.25% trypsin for 1 h at 37°C. The cells were lysed by passage through a 1-ml syringe with a 25-gauge needle 40 times. Cell lysates were incubated for 1 h at 37°C once more, the trypsin was inactivated, and the samples were analyzed by Western blot analysis with a cocktail of HPV16 L1-specific mouse MAbs (IID5, 33L1-7, and 312F).
    Figure Legend Snippet: Some full-length L1 protein accompanies the viral genome to the nucleus. (A) HeLa cells were infected with HPV16 pseudovirus with or without 1.5 μM Eg5 inhibitor III (Eg5i) and tracked via live-cell imaging using the IncuCyte Zoom for 48 h. Note that the images are depicted at 18 hpi. (B) HaCaT cells were infected with EdU-labeled HPV16 pseudovirus in the presence of 1.5 μM Eg5i. The cells were fixed at 24 hpi and permeabilized with either digitonin at 0.625 μg/ml or 0.5% TX-100 and then treated with AF555 (green) in Click-iT reaction buffer. The cells were permeabilized again with 0.5% TX-100 and treated with AF647 (red) in Click-iT reaction buffer. Lastly, the cells were mounted in DAPI (blue). (C) HaCaT cells were infected with EdU-labeled pseudovirus and treated with 1.5 μM Eg5i. At 24 hpi, the cells were fixed and permeabilized in 0.5% TX-100. Next, the cells were treated with AF555 (red) in Click-iT reaction buffer, followed by incubation with AF488-conjugated anti-α-tubulin (white) and MAb 33L1-7 (green). Lastly, the cells were mounted in DAPI (blue). Note the colocalization between EdU and L1 signal denoted by white arrows. (D) HeLa cells were infected with HPV16 pseudovirus in the presence of 1.5 μM Eg5i. Cells were trypsinized for 2 min, and monoastral cells were collected. Next, the cells were treated with 15 μl of 0.25% trypsin for 1 h at 37°C. The cells were lysed by passage through a 1-ml syringe with a 25-gauge needle 40 times. Cell lysates were incubated for 1 h at 37°C once more, the trypsin was inactivated, and the samples were analyzed by Western blot analysis with a cocktail of HPV16 L1-specific mouse MAbs (IID5, 33L1-7, and 312F).

    Techniques Used: Infection, Live Cell Imaging, Labeling, Incubation, Western Blot

    L1 proteins that accompany the viral genome into the nucleus dissociate after release of the viral genome. (A) At 24 h, uninfected HaCaT cells were fixed, permeabilized with either digitonin at 0.625 μg/ml or 0.5% TX-100, and treated with AF555 (green) in Click-iT reaction buffer. Next, the cells were incubated with pAb rabbit anti-TGN46 (cyan), which recognizes a luminal epitope of TGN46. Lastly, the cells were permeabilized in 0.5% TX-100, followed by incubation with goat anti-rabbit secondary antibody and subsequent mounting in DAPI (white). Note the lack of reactivity of luminal anti-TGN46 antibody after digitonin treatment. (B and C) At 24 hpi, HaCaT cells infected with EdU-labeled pseudovirus were fixed, permeabilized with digitonin at 0.625 μg/ml (B) or 0.5% TX-100 (C), and treated with AF555 (green) in Click-iT reaction buffer. The cells were permeabilized again with 0.5% TX-100 and treated with AF647 (red) in Click-iT reaction buffer. Lastly, the cells were incubated with mouse MAb 33L1-7 (cyan) for specific detection of the L1 protein and mounted in DAPI (white). (D and E) The percent accessibility of viral genome was determined by counting the number of red-only (inaccessible [IN]) or red/green (accessible (AC) stained EdU puncta associated with condensed chromosomes on mitotic cells or nuclear localized in interphase cells. Colocalization of L1 and EdU puncta was quantified by counting the number of EdU puncta that colocalized with L1 signal. Quantifications are from two repeat experiments analyzing two to three Z-stack images per cell ( n = 30 to 40 cells, and > 800 EdU puncta were counted per experiment). Mitosis: %IN = 88.58% ± 7.67%, %AC = 11.42% ± 7.67%, %L1 of IN = 82.3% ± 7.30%, %L1 of AC = 42.665% ± 9.335%. Interphase: %IN = 34.53% ± 0.427%, %AC = 68.36% ± 6.463%, %L1 of IN = 58.8% ± 3.805%, %L1 of AC = 20.84% ± 9.159%.
    Figure Legend Snippet: L1 proteins that accompany the viral genome into the nucleus dissociate after release of the viral genome. (A) At 24 h, uninfected HaCaT cells were fixed, permeabilized with either digitonin at 0.625 μg/ml or 0.5% TX-100, and treated with AF555 (green) in Click-iT reaction buffer. Next, the cells were incubated with pAb rabbit anti-TGN46 (cyan), which recognizes a luminal epitope of TGN46. Lastly, the cells were permeabilized in 0.5% TX-100, followed by incubation with goat anti-rabbit secondary antibody and subsequent mounting in DAPI (white). Note the lack of reactivity of luminal anti-TGN46 antibody after digitonin treatment. (B and C) At 24 hpi, HaCaT cells infected with EdU-labeled pseudovirus were fixed, permeabilized with digitonin at 0.625 μg/ml (B) or 0.5% TX-100 (C), and treated with AF555 (green) in Click-iT reaction buffer. The cells were permeabilized again with 0.5% TX-100 and treated with AF647 (red) in Click-iT reaction buffer. Lastly, the cells were incubated with mouse MAb 33L1-7 (cyan) for specific detection of the L1 protein and mounted in DAPI (white). (D and E) The percent accessibility of viral genome was determined by counting the number of red-only (inaccessible [IN]) or red/green (accessible (AC) stained EdU puncta associated with condensed chromosomes on mitotic cells or nuclear localized in interphase cells. Colocalization of L1 and EdU puncta was quantified by counting the number of EdU puncta that colocalized with L1 signal. Quantifications are from two repeat experiments analyzing two to three Z-stack images per cell ( n = 30 to 40 cells, and > 800 EdU puncta were counted per experiment). Mitosis: %IN = 88.58% ± 7.67%, %AC = 11.42% ± 7.67%, %L1 of IN = 82.3% ± 7.30%, %L1 of AC = 42.665% ± 9.335%. Interphase: %IN = 34.53% ± 0.427%, %AC = 68.36% ± 6.463%, %L1 of IN = 58.8% ± 3.805%, %L1 of AC = 20.84% ± 9.159%.

    Techniques Used: Incubation, Infection, Labeling, Staining

    L2 proteins that accompany the viral genome into the nucleus remain associated with the viral genome. (A and B) At 24 hpi, HaCaT cells infected with EdU-labeled pseudovirus were fixed, permeabilized with digitonin at 0.625 μg/ml of (A) or 0.5% TX-100 (B), and treated with AF555 (green) in Click-iT reaction buffer. The cells were permeabilized again with 0.5% TX-100 and treated with AF647 (red) in Click-iT reaction buffer. Lastly, the cells were incubated with mouse MAb 33L2-1 (cyan) for specific detection of the L2 protein and mounted in DAPI (white). (C and D) The percent accessibility of the viral genome was determined by counting the number of red-only (inaccessible [IN]) or red/green (accessible [AC]) stained EdU puncta associated with condensed chromosomes on mitotic cells or nuclear localized in interphase cells. Colocalization of L2 and EdU puncta was quantified by counting the number of EdU puncta that colocalized with L2 signal. Quantifications are from two repeat experiments analyzing two to three Z-stack images per cell ( n = 30 to 40 cells and > 800 EdU puncta counter per experiment). Mitosis: %IN = 83.67% ± 8.435%, %AC = 16.34% ± 8.435%, %L2 of IN = 46.15% ± 6.15%, %L2 of AC = 24.45% ± 16.55%. Interphase: %IN = 42.15% ± 1.35%, %AC = 57.85% ± 1.35%, %L2 of IN = 44.65% ± 6.35%, %L2 of AC = 30.65% ± 3.35%.
    Figure Legend Snippet: L2 proteins that accompany the viral genome into the nucleus remain associated with the viral genome. (A and B) At 24 hpi, HaCaT cells infected with EdU-labeled pseudovirus were fixed, permeabilized with digitonin at 0.625 μg/ml of (A) or 0.5% TX-100 (B), and treated with AF555 (green) in Click-iT reaction buffer. The cells were permeabilized again with 0.5% TX-100 and treated with AF647 (red) in Click-iT reaction buffer. Lastly, the cells were incubated with mouse MAb 33L2-1 (cyan) for specific detection of the L2 protein and mounted in DAPI (white). (C and D) The percent accessibility of the viral genome was determined by counting the number of red-only (inaccessible [IN]) or red/green (accessible [AC]) stained EdU puncta associated with condensed chromosomes on mitotic cells or nuclear localized in interphase cells. Colocalization of L2 and EdU puncta was quantified by counting the number of EdU puncta that colocalized with L2 signal. Quantifications are from two repeat experiments analyzing two to three Z-stack images per cell ( n = 30 to 40 cells and > 800 EdU puncta counter per experiment). Mitosis: %IN = 83.67% ± 8.435%, %AC = 16.34% ± 8.435%, %L2 of IN = 46.15% ± 6.15%, %L2 of AC = 24.45% ± 16.55%. Interphase: %IN = 42.15% ± 1.35%, %AC = 57.85% ± 1.35%, %L2 of IN = 44.65% ± 6.35%, %L2 of AC = 30.65% ± 3.35%.

    Techniques Used: Infection, Labeling, Incubation, Staining

    32) Product Images from "Expression and regulation of avian beta-defensin 8 protein in immune tissues and cell lines of chickens"

    Article Title: Expression and regulation of avian beta-defensin 8 protein in immune tissues and cell lines of chickens

    Journal: Asian-Australasian Journal of Animal Sciences

    doi: 10.5713/ajas.17.0836

    Determination of AvBD8 protein expression in immune cell lines by performing immunocytochemical analysis. Cultured chicken CU91 T-, DT40 B-, HD11 macrophage-, and OU2 fibroblast-cell lines were incubated with the rabbit anti-AvBD8 primary antibody, followed by incubation with the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody, and were counterstained with 4′,6-diamidino-2-phenylindole. Control cells were incubated with secondary antibody only. AvBD8, avian beta-defensin 8; IgG, immunoglobulin G. Scale bar: 20 μm.
    Figure Legend Snippet: Determination of AvBD8 protein expression in immune cell lines by performing immunocytochemical analysis. Cultured chicken CU91 T-, DT40 B-, HD11 macrophage-, and OU2 fibroblast-cell lines were incubated with the rabbit anti-AvBD8 primary antibody, followed by incubation with the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody, and were counterstained with 4′,6-diamidino-2-phenylindole. Control cells were incubated with secondary antibody only. AvBD8, avian beta-defensin 8; IgG, immunoglobulin G. Scale bar: 20 μm.

    Techniques Used: Expressing, Cell Culture, Incubation

    Determination of AvBD8 protein expression in immune tissues of male WL chickens by performing immunohistochemical analysis. Immunohistochemical analysis was performed to assess AvBD8 protein expression in the thymus, spleen, liver, small intestine, and ceca of male WL chickens aged 25 weeks. Frozen sections were incubated with the rabbit anti-AvBD8 primary antibody, followed by incubation with the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody, and were counterstained with 4′,6-diamidino-2-phenylindole. Control sections were incubated with secondary antibody only. Boxed region in the middle column is enlarged in the right column. AvBD8, avian beta-defensin 8; WL, White Leghorn; IgG, immunoglobulin G. Scale bar: 200 μm (left and middle columns) and 50 μm (right column). Arrowheads indicate strong AvBD8 signal in the intestinal mucosal layer.
    Figure Legend Snippet: Determination of AvBD8 protein expression in immune tissues of male WL chickens by performing immunohistochemical analysis. Immunohistochemical analysis was performed to assess AvBD8 protein expression in the thymus, spleen, liver, small intestine, and ceca of male WL chickens aged 25 weeks. Frozen sections were incubated with the rabbit anti-AvBD8 primary antibody, followed by incubation with the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody, and were counterstained with 4′,6-diamidino-2-phenylindole. Control sections were incubated with secondary antibody only. Boxed region in the middle column is enlarged in the right column. AvBD8, avian beta-defensin 8; WL, White Leghorn; IgG, immunoglobulin G. Scale bar: 200 μm (left and middle columns) and 50 μm (right column). Arrowheads indicate strong AvBD8 signal in the intestinal mucosal layer.

    Techniques Used: Expressing, Immunohistochemistry, Incubation

    33) Product Images from "Distinct and Shared Roles of ?-Arrestin-1 and ?-Arrestin-2 on the Regulation of C3a Receptor Signaling in Human Mast Cells"

    Article Title: Distinct and Shared Roles of ?-Arrestin-1 and ?-Arrestin-2 on the Regulation of C3a Receptor Signaling in Human Mast Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0019585

    C3a-induced ERK1/2 phosphorylation is enhanced in β-arrestin-1, β-arrestin-2 and double KD cells. shRNA control or β-arrestin KD HMC-1 cells (1×10 6 /ml) were exposed to C3a (100 nM) for 1, 5 and 10, 15 and 30 min. Cell lysates were separated on SDS-PAGE and blots were probed with anti-phospho-ERK1/2 antibody followed by anti-rabbit IgG-HRP. The blots were then stripped and reprobed with anti-ERK1/2 antibody followed by anti-rabbit IgG-HRP. Immunoreactive band were visualized by SuperSignal West Femto maximum sensitivity substrate. (A) Representative immunoblots from three similar experiments are shown. (B) ERK1/2 phosphorylation was quantified using Image J as shown in the line graph. Data represent the mean ± SEM from three independent experiments. Statistical significance was determined by two way ANOVA with Bonferroni's post test. ** indicates p
    Figure Legend Snippet: C3a-induced ERK1/2 phosphorylation is enhanced in β-arrestin-1, β-arrestin-2 and double KD cells. shRNA control or β-arrestin KD HMC-1 cells (1×10 6 /ml) were exposed to C3a (100 nM) for 1, 5 and 10, 15 and 30 min. Cell lysates were separated on SDS-PAGE and blots were probed with anti-phospho-ERK1/2 antibody followed by anti-rabbit IgG-HRP. The blots were then stripped and reprobed with anti-ERK1/2 antibody followed by anti-rabbit IgG-HRP. Immunoreactive band were visualized by SuperSignal West Femto maximum sensitivity substrate. (A) Representative immunoblots from three similar experiments are shown. (B) ERK1/2 phosphorylation was quantified using Image J as shown in the line graph. Data represent the mean ± SEM from three independent experiments. Statistical significance was determined by two way ANOVA with Bonferroni's post test. ** indicates p

    Techniques Used: shRNA, SDS Page, Western Blot

    34) Product Images from "The microvascular niche instructs T cells in large vessel vasculitis via the VEGF-Jagged1-Notch pathway"

    Article Title: The microvascular niche instructs T cells in large vessel vasculitis via the VEGF-Jagged1-Notch pathway

    Journal: Science translational medicine

    doi: 10.1126/scitranslmed.aal3322

    Adventitial mvECs in GCA arteries express Jagged1 Tissue biopsies were collected from temporal arteries and from aortic wall specimens. Arteries affected by GCA had typical transmural granulomatous arteritis (GCA-positive and GCA aortitis). Temporal arteries with no inflammatory infiltrates (GCA-negative) served as controls. Nuclei were marked with 4´,6-diamidino-2-phenylindole. Scale bars 50 μm. (A) Tissue-infiltrating T cells were identified by staining sections from GCA-negative and GCA-positive temporal arteries and from GCA aortitis with mouse anti-human CD3 antibody. Antibody binding was visualized with Alexa Fluor 594-labeled anti-mouse immunoglobulin G (IgG) secondary antibody (red). Representative stains from eight samples each are shown here. (B) mRNA was extracted from GCA-positive and GCA-negative temporal arteries and analyzed by reverse transcription polymerase chain reaction (RT-PCR) for the expression of JAG1 , DLL1 and DLL4 transcripts. Results (mean ± SEM) from six GCA arteries and six healthy arteries are shown. N.s., not significant. (C and D) Dual-color staining was applied to identify Jagged1 and Delta-like 1 expressed on endothelial cells in GCA-positive, GCA aortitis, and GCA-negative arteries. Tissue sections were double-stained with mouse anti-human CD31 antibody and rabbit anti-human Jagged1 antibody or rabbit anti-human Delta-like 1 antibody. Alexa Fluor 594 anti-mouse IgG (red) and Alexa Fluor 488 anti-rabbit IgG (green) were used as secondary antibodies. Merged images demonstrate colocalization of both markers (yellow). Representative images from eight samples each.
    Figure Legend Snippet: Adventitial mvECs in GCA arteries express Jagged1 Tissue biopsies were collected from temporal arteries and from aortic wall specimens. Arteries affected by GCA had typical transmural granulomatous arteritis (GCA-positive and GCA aortitis). Temporal arteries with no inflammatory infiltrates (GCA-negative) served as controls. Nuclei were marked with 4´,6-diamidino-2-phenylindole. Scale bars 50 μm. (A) Tissue-infiltrating T cells were identified by staining sections from GCA-negative and GCA-positive temporal arteries and from GCA aortitis with mouse anti-human CD3 antibody. Antibody binding was visualized with Alexa Fluor 594-labeled anti-mouse immunoglobulin G (IgG) secondary antibody (red). Representative stains from eight samples each are shown here. (B) mRNA was extracted from GCA-positive and GCA-negative temporal arteries and analyzed by reverse transcription polymerase chain reaction (RT-PCR) for the expression of JAG1 , DLL1 and DLL4 transcripts. Results (mean ± SEM) from six GCA arteries and six healthy arteries are shown. N.s., not significant. (C and D) Dual-color staining was applied to identify Jagged1 and Delta-like 1 expressed on endothelial cells in GCA-positive, GCA aortitis, and GCA-negative arteries. Tissue sections were double-stained with mouse anti-human CD31 antibody and rabbit anti-human Jagged1 antibody or rabbit anti-human Delta-like 1 antibody. Alexa Fluor 594 anti-mouse IgG (red) and Alexa Fluor 488 anti-rabbit IgG (green) were used as secondary antibodies. Merged images demonstrate colocalization of both markers (yellow). Representative images from eight samples each.

    Techniques Used: Staining, Binding Assay, Labeling, Reverse Transcription Polymerase Chain Reaction, Expressing

    35) Product Images from "Interleukin-like EMT inducer (ILEI) promotes melanoma invasiveness and is transcriptionally up-regulated by upstream stimulatory factor-1 (USF-1)"

    Article Title: Interleukin-like EMT inducer (ILEI) promotes melanoma invasiveness and is transcriptionally up-regulated by upstream stimulatory factor-1 (USF-1)

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.003616

    USF-1 interacts with the ILEI promoter sequence. A, 5′-biotin–tagged ILEI promoter oligonucleotide constructs with WT or mutant E-box. Biotin pulldown analysis of nuclear extracts from 501-Mel melanoma cells, followed by immunoblot for USF-1. B, PCR primers flanking the ILEI promoter E-box used in ChIP analysis. ChIP analysis of 501-Mel melanoma cell lines immunoprecipitated with control IgG or USF-1 antibody. PCR analysis conducted with primers targeting FAM3C, TYR, or HO1 promoter.
    Figure Legend Snippet: USF-1 interacts with the ILEI promoter sequence. A, 5′-biotin–tagged ILEI promoter oligonucleotide constructs with WT or mutant E-box. Biotin pulldown analysis of nuclear extracts from 501-Mel melanoma cells, followed by immunoblot for USF-1. B, PCR primers flanking the ILEI promoter E-box used in ChIP analysis. ChIP analysis of 501-Mel melanoma cell lines immunoprecipitated with control IgG or USF-1 antibody. PCR analysis conducted with primers targeting FAM3C, TYR, or HO1 promoter.

    Techniques Used: Sequencing, Construct, Mutagenesis, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Immunoprecipitation

    36) Product Images from "A Stem-Cell-Derived Platform Enables Complete Cryptosporidium Development In Vitro and Genetic Tractability"

    Article Title: A Stem-Cell-Derived Platform Enables Complete Cryptosporidium Development In Vitro and Genetic Tractability

    Journal: Cell Host & Microbe

    doi: 10.1016/j.chom.2019.05.007

    In vitro production of oocysts in ALI culture (A) Effect of filtration on removal of residual C. parvum oocysts. Oocysts were excysted, filtered using the indicated pore sizes, added to PLL-coated coverslips, and stained with Crypt-a-glo directly conjugated to FITC. The number of residual oocysts found in filtered samples were counted from replicate 63× fields from a representative experiment. (B) Detection of C. parvum oocysts in ALI cultures. Transwells were infected on day 3 post top medium removal with 1 μm-filtered sporozoites. On specified days post infection, transwells were fixed and stained with Crypt-a-glo directly conjugated to FITC and Pan Cp, detected with goat anti-rabbit IgG Alexa Fluor 568. Oocyst numbers per 63× field were counted for three independent experiments (Exp A, Exp B, Exp C). The combined mean of each time point was compared to the combined mean of day 1 using a two-way ANOVA corrected for multiple comparisons using the Dunnett method, ∗∗∗∗ p
    Figure Legend Snippet: In vitro production of oocysts in ALI culture (A) Effect of filtration on removal of residual C. parvum oocysts. Oocysts were excysted, filtered using the indicated pore sizes, added to PLL-coated coverslips, and stained with Crypt-a-glo directly conjugated to FITC. The number of residual oocysts found in filtered samples were counted from replicate 63× fields from a representative experiment. (B) Detection of C. parvum oocysts in ALI cultures. Transwells were infected on day 3 post top medium removal with 1 μm-filtered sporozoites. On specified days post infection, transwells were fixed and stained with Crypt-a-glo directly conjugated to FITC and Pan Cp, detected with goat anti-rabbit IgG Alexa Fluor 568. Oocyst numbers per 63× field were counted for three independent experiments (Exp A, Exp B, Exp C). The combined mean of each time point was compared to the combined mean of day 1 using a two-way ANOVA corrected for multiple comparisons using the Dunnett method, ∗∗∗∗ p

    Techniques Used: In Vitro, Filtration, Staining, Infection

    ALI Transwells support genetic crosses of C. parvum in vitro (A) Diagram of targeting construct designed to replace the endogenous tk locus (cgd5_4440) with GFP and Nluc-P2A-Neo R cassette. (B) Relative luminescence normalized to total number of parasites in ALI transwells at 1 and 4 days post infection (dpi). Transwells were infected with ∼1 × 10 7 unfiltered sporozoites that were electroporated with the TK-GFP-Nluc-P2A-neo-TK reporter and a Cas9 plasmid with a TK gRNA. Transwells were cultured in medium containing PBS (light green) as a control or 20 mM paromomycin (PRM, dark green). Data plotted as mean ± S.D. from two transwells per time point from a representative experiment. Nonsignificant (p = 0.11), unpaired Student’s t test between PBS and PRM-treated transwells 4 dpi. (C) Image of whole-mount ALI transwells 5 dpi with transfected C. parvum from same experiment as (B) stained with anti-GFP followed by goat anti-rabbit IgG Alexa Fluor 488. Scale bar, 10 μm. (D) Merged image of (C) with a Pan Cp polyclonal antibody, which recognizes all C. parvum stages, followed by goat antirat IgG Alexa Fluor 568. Scale bar, 10 μm. (E) Diagram of targeting construct designed to replace the endogenous uprt locus (cgd1_1900) with mCherry and Nluc-P2A-Neo R cassette as (A). (F) Relative luminescence normalized to total number of parasites in ALI transwells at 1 and 3 dpi. Transwells were infected with ∼1 × 10 7 unfiltered sporozoites per transwell that were electroporated with the UPRT-mCh-Nluc-P2A-neo-UPRT reporter and a Cas9 plasmid with a UPRT gRNA. Transwells were cultured in medium containing PBS (pink) as a control or 20 mM PRM (red). Data plotted as mean ± S.D. from two transwells per time point from a representative experiment. ∗ p
    Figure Legend Snippet: ALI Transwells support genetic crosses of C. parvum in vitro (A) Diagram of targeting construct designed to replace the endogenous tk locus (cgd5_4440) with GFP and Nluc-P2A-Neo R cassette. (B) Relative luminescence normalized to total number of parasites in ALI transwells at 1 and 4 days post infection (dpi). Transwells were infected with ∼1 × 10 7 unfiltered sporozoites that were electroporated with the TK-GFP-Nluc-P2A-neo-TK reporter and a Cas9 plasmid with a TK gRNA. Transwells were cultured in medium containing PBS (light green) as a control or 20 mM paromomycin (PRM, dark green). Data plotted as mean ± S.D. from two transwells per time point from a representative experiment. Nonsignificant (p = 0.11), unpaired Student’s t test between PBS and PRM-treated transwells 4 dpi. (C) Image of whole-mount ALI transwells 5 dpi with transfected C. parvum from same experiment as (B) stained with anti-GFP followed by goat anti-rabbit IgG Alexa Fluor 488. Scale bar, 10 μm. (D) Merged image of (C) with a Pan Cp polyclonal antibody, which recognizes all C. parvum stages, followed by goat antirat IgG Alexa Fluor 568. Scale bar, 10 μm. (E) Diagram of targeting construct designed to replace the endogenous uprt locus (cgd1_1900) with mCherry and Nluc-P2A-Neo R cassette as (A). (F) Relative luminescence normalized to total number of parasites in ALI transwells at 1 and 3 dpi. Transwells were infected with ∼1 × 10 7 unfiltered sporozoites per transwell that were electroporated with the UPRT-mCh-Nluc-P2A-neo-UPRT reporter and a Cas9 plasmid with a UPRT gRNA. Transwells were cultured in medium containing PBS (pink) as a control or 20 mM PRM (red). Data plotted as mean ± S.D. from two transwells per time point from a representative experiment. ∗ p

    Techniques Used: In Vitro, Construct, Infection, Plasmid Preparation, Cell Culture, Transfection, Staining

    Development of an In Vitro System for Cultivation of C. Parvum (A) Model of air-liquid interface (ALI) culture method. See methods for details and Figures S1 A and S1B for further description. (B) Histological examination of ALI cultures. Sections of ALI 3 days post-infection (PI) stained with hematoxylin and eosin (H E), or rabbit pAb to detect C. parvum (referred to as Pan Cp) using immunohistochemistry (IHC). White arrows in middle panel highlight C. parvum . Scale bar, 20 μm. (C) Growth of C. parvum in ALI cultures infected 3 days post top medium removal with 2 × 10 5 unfiltered C. parvum oocysts. The graph depicts qPCR measurement of C. parvum and mouse GAPDH equivalents (eq). Means ± S.D. of two transwells per time point from a representative experiment. See Figure S1 C for the replicate experiment. (D) Detection of developmental stages of C. parvum in ALI cultures. Infected ALI transwells were fixed and stained with specified mouse mAbs (i.e., 1B5, 1A5) followed by goat antimouse IgG Alexa Fluor 488, Crypt-a-glo directly conjugated to FITC, or Pan Cp followed by goat anti-rabbit IgG Alexa Fluor 568. Hoechst staining for DNA. Scale bar, 3 μm. See also Figure S2 .
    Figure Legend Snippet: Development of an In Vitro System for Cultivation of C. Parvum (A) Model of air-liquid interface (ALI) culture method. See methods for details and Figures S1 A and S1B for further description. (B) Histological examination of ALI cultures. Sections of ALI 3 days post-infection (PI) stained with hematoxylin and eosin (H E), or rabbit pAb to detect C. parvum (referred to as Pan Cp) using immunohistochemistry (IHC). White arrows in middle panel highlight C. parvum . Scale bar, 20 μm. (C) Growth of C. parvum in ALI cultures infected 3 days post top medium removal with 2 × 10 5 unfiltered C. parvum oocysts. The graph depicts qPCR measurement of C. parvum and mouse GAPDH equivalents (eq). Means ± S.D. of two transwells per time point from a representative experiment. See Figure S1 C for the replicate experiment. (D) Detection of developmental stages of C. parvum in ALI cultures. Infected ALI transwells were fixed and stained with specified mouse mAbs (i.e., 1B5, 1A5) followed by goat antimouse IgG Alexa Fluor 488, Crypt-a-glo directly conjugated to FITC, or Pan Cp followed by goat anti-rabbit IgG Alexa Fluor 568. Hoechst staining for DNA. Scale bar, 3 μm. See also Figure S2 .

    Techniques Used: In Vitro, Infection, Staining, Immunohistochemistry, Real-time Polymerase Chain Reaction

    Infectivity of oocysts produced in ALI cultures (A) Detection of oocysts in ALI cultures treated with bleach. ALI transwell cultures were infected with 1 μm-filtered sporozoites. On days 1 and 3 postinfection, transwells were bleached, washed, pelleted, adhered to PLL-coated coverslips, and stained with Pan Cp followed by goat anti-rabbit Alexa Fluor 568 and Crypt-a-glo directly conjugated to FITC. Each data point is the number of oocysts in a single 63X field from a single representative experiment. Data were analyzed using a Mann-Whitney U test. ∗∗∗∗ p
    Figure Legend Snippet: Infectivity of oocysts produced in ALI cultures (A) Detection of oocysts in ALI cultures treated with bleach. ALI transwell cultures were infected with 1 μm-filtered sporozoites. On days 1 and 3 postinfection, transwells were bleached, washed, pelleted, adhered to PLL-coated coverslips, and stained with Pan Cp followed by goat anti-rabbit Alexa Fluor 568 and Crypt-a-glo directly conjugated to FITC. Each data point is the number of oocysts in a single 63X field from a single representative experiment. Data were analyzed using a Mann-Whitney U test. ∗∗∗∗ p

    Techniques Used: Infection, Produced, Staining, MANN-WHITNEY

    37) Product Images from "Upregulation of glycosaminoglycan synthesis by Neurotropin in nucleus pulposus cells via stimulation of chondroitin sulfate N-acetylgalactosaminyltransferase 1: A new approach to attenuation of intervertebral disc degeneration"

    Article Title: Upregulation of glycosaminoglycan synthesis by Neurotropin in nucleus pulposus cells via stimulation of chondroitin sulfate N-acetylgalactosaminyltransferase 1: A new approach to attenuation of intervertebral disc degeneration

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0202640

    NTP activates AKT phosphorylation. Phosphorylation of AKT by NTP was examined in NP cells by staining with specific antibody against pAKT (Ser473). A) Time courses show the integrated signals of the Alexa Fluor 488-labeled pAKT (shown in green in the photograph) indicated peaks from 15 to 90 minutes after addition of NTP and AsAP. The peak levels of phosphorylation were much higher in the samples co-treated with 0.1, 1.0 and 10 mNU/ml NTP and AsAP than in those treated with AsAP only. Capture of immunofluorescence images and integration of the signal intensity in the cultured cells were performed using the ArrayScan system. Every serial sample was assayed in duplicated wells (Donor, N = 1). Red arrow shows the time point at which the representative photographs were taken. Nuclei are shown in blue by staining with DAPI. Bars are 100 μm. B) Western blot analysis of phosphorylation of AKT using the same antibody. Thirty minutes and 60 minutes after addition of NTP, cells were harvested and whole cell lysate was immunoblotted (N = 2). Addition of 0.1 mNU/ml NTP upregulated phosphorylation of AKT (Ser473) compared with control, but higher concentrations of NTP (1.0 and 10 mNU/ml) were not proportionally more effective (left and right). Addition of AsAP alone was also effective on AKT phosphorylation (right). AKT is constant under all culture conditions.
    Figure Legend Snippet: NTP activates AKT phosphorylation. Phosphorylation of AKT by NTP was examined in NP cells by staining with specific antibody against pAKT (Ser473). A) Time courses show the integrated signals of the Alexa Fluor 488-labeled pAKT (shown in green in the photograph) indicated peaks from 15 to 90 minutes after addition of NTP and AsAP. The peak levels of phosphorylation were much higher in the samples co-treated with 0.1, 1.0 and 10 mNU/ml NTP and AsAP than in those treated with AsAP only. Capture of immunofluorescence images and integration of the signal intensity in the cultured cells were performed using the ArrayScan system. Every serial sample was assayed in duplicated wells (Donor, N = 1). Red arrow shows the time point at which the representative photographs were taken. Nuclei are shown in blue by staining with DAPI. Bars are 100 μm. B) Western blot analysis of phosphorylation of AKT using the same antibody. Thirty minutes and 60 minutes after addition of NTP, cells were harvested and whole cell lysate was immunoblotted (N = 2). Addition of 0.1 mNU/ml NTP upregulated phosphorylation of AKT (Ser473) compared with control, but higher concentrations of NTP (1.0 and 10 mNU/ml) were not proportionally more effective (left and right). Addition of AsAP alone was also effective on AKT phosphorylation (right). AKT is constant under all culture conditions.

    Techniques Used: Staining, Labeling, Immunofluorescence, Cell Culture, Western Blot

    38) Product Images from "Humoral response in experimental autoimmune encephalomyelitis targets neural precursor cells in the central nervous system of naive rodents"

    Article Title: Humoral response in experimental autoimmune encephalomyelitis targets neural precursor cells in the central nervous system of naive rodents

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-017-0995-2

    MOG 35–55 -EAE induction elicits a humoral response directed towards the spinal cord and NPCs. a Mean clinical score of all animals during EAE course. Error bars show the standard statistical error of the mean (SEM). b XTT assay indicated the relative NPC viability % of NPCs treated with purified IgG from EAE-AS and unpurified EAE-AS compared with NPCs treated with NAIVE-AS (control), in three different concentrations (0.1, 1, and 10 μg/ml). Data are presented as relative viability percentage (%) as mean ± SEM. Western blot of various antisera from animals immunized with MOG (EAE-AS) yielded one band approximately at 30 kDa on spinal cord substrate ( c ) and bands at above 60 kDa, above 40 kDa, and around 30 kDa on NPC substrate ( d ). Lane probed with EAE-AS demonstrates a representative antiserum. Anti-MOG antibody and anti-actin-loading control were also used
    Figure Legend Snippet: MOG 35–55 -EAE induction elicits a humoral response directed towards the spinal cord and NPCs. a Mean clinical score of all animals during EAE course. Error bars show the standard statistical error of the mean (SEM). b XTT assay indicated the relative NPC viability % of NPCs treated with purified IgG from EAE-AS and unpurified EAE-AS compared with NPCs treated with NAIVE-AS (control), in three different concentrations (0.1, 1, and 10 μg/ml). Data are presented as relative viability percentage (%) as mean ± SEM. Western blot of various antisera from animals immunized with MOG (EAE-AS) yielded one band approximately at 30 kDa on spinal cord substrate ( c ) and bands at above 60 kDa, above 40 kDa, and around 30 kDa on NPC substrate ( d ). Lane probed with EAE-AS demonstrates a representative antiserum. Anti-MOG antibody and anti-actin-loading control were also used

    Techniques Used: XTT Assay, Purification, Western Blot

    39) Product Images from "Argonaute‐2 Promotes miR‐18a Entry in Human Brain Endothelial Cells"

    Article Title: Argonaute‐2 Promotes miR‐18a Entry in Human Brain Endothelial Cells

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    doi: 10.1161/JAHA.114.000968

    Facilitated transport is involved in miR‐18a delivery. A, AVM‐BEC and control BEC were treated with AVM‐BEC‐CM plus miR‐18a (40 nmol/L) at 4°C and 37°C for 30 minutes. Intracellular miR‐18a was measured using qPCR as described previously, showing that at 4°C miRNA entry was only minimally compromised (n=3). B, The distribution of Ago‐2 (red) was identified using immunocytochemistry. At 4°C untreated AVM‐BEC expressed high levels of intracellular Ago‐2 (i) compared to untreated control BEC (ii). When control BEC were treated with AVM‐BEC‐CM plus miR‐18a at 4°C, Ago‐2 staining was apparent and associated with the cell membrane (iii; white arrows) (n=3). Blue staining denotes nuclear staining. C, The formation of a ribonucleoprotein complex between Ago‐2 and miR‐18a was determined by immunoprecipitation and immunoblotting (left panel) and qPCR (right panel). Ago‐2 was detected only in the 2 fractions in contact with anti‐Ago‐2, as expected. Only the fraction with both Ago‐2 and miR‐18, but not miR‐18a alone, led to the detection of miR‐18a by qPCR. Rabbit IgG served as the isotypic control. D, GW4869 (10 to 50 μmol/L), a specific inhibitor of N‐Smase‐2 (neutral sphingomyelinase‐2) did not interfere with miR‐18a uptake in AVM‐BEC. Data are presented as mean±standard error of the mean (SEM). Each human specimen‐derived cell culture represents a unit of analysis (n). AVM indicates arteriovenous malformation; Ago‐2, Argonaute‐2; BEC, brain endothelial cells; CM, conditioned media; qPCR, quantitative real‐time polymerase chain reaction.
    Figure Legend Snippet: Facilitated transport is involved in miR‐18a delivery. A, AVM‐BEC and control BEC were treated with AVM‐BEC‐CM plus miR‐18a (40 nmol/L) at 4°C and 37°C for 30 minutes. Intracellular miR‐18a was measured using qPCR as described previously, showing that at 4°C miRNA entry was only minimally compromised (n=3). B, The distribution of Ago‐2 (red) was identified using immunocytochemistry. At 4°C untreated AVM‐BEC expressed high levels of intracellular Ago‐2 (i) compared to untreated control BEC (ii). When control BEC were treated with AVM‐BEC‐CM plus miR‐18a at 4°C, Ago‐2 staining was apparent and associated with the cell membrane (iii; white arrows) (n=3). Blue staining denotes nuclear staining. C, The formation of a ribonucleoprotein complex between Ago‐2 and miR‐18a was determined by immunoprecipitation and immunoblotting (left panel) and qPCR (right panel). Ago‐2 was detected only in the 2 fractions in contact with anti‐Ago‐2, as expected. Only the fraction with both Ago‐2 and miR‐18, but not miR‐18a alone, led to the detection of miR‐18a by qPCR. Rabbit IgG served as the isotypic control. D, GW4869 (10 to 50 μmol/L), a specific inhibitor of N‐Smase‐2 (neutral sphingomyelinase‐2) did not interfere with miR‐18a uptake in AVM‐BEC. Data are presented as mean±standard error of the mean (SEM). Each human specimen‐derived cell culture represents a unit of analysis (n). AVM indicates arteriovenous malformation; Ago‐2, Argonaute‐2; BEC, brain endothelial cells; CM, conditioned media; qPCR, quantitative real‐time polymerase chain reaction.

    Techniques Used: Real-time Polymerase Chain Reaction, Immunocytochemistry, Staining, Immunoprecipitation, Derivative Assay, Cell Culture

    40) Product Images from "The sickle cell trait affects contact dynamics and endothelial cell activation in Plasmodium falciparum-infected erythrocytes"

    Article Title: The sickle cell trait affects contact dynamics and endothelial cell activation in Plasmodium falciparum-infected erythrocytes

    Journal: Communications Biology

    doi: 10.1038/s42003-018-0223-3

    Reduced endothelial cell activation by infected HbAS erythrocytes. a Representative confocal microscopy images showing the subcellular localization of p65-NFĸB subunit in human dermal microvascular endothelial cells (HDMEC) upon TNF-α treatment (100 units ml −1 for 2 h) or upon co-culture with parasitized HbAA or HbAS erythrocytes (1 × 10 7 infected red blood cells at the trophozoite stage for 2 h) under static conditions. Medium served as a negative control. The p65-NFĸB subunit was localized using a specific polyclonal rabbit antiserum (dilution 1:200) and an Alexa-488 conjugated goat anti-rabbit IgG antiserum (1:400) as secondary antibody. The nuclei were stained using Hoechst 33342. Scale bar, 20 µm. b Percentage of HDMECs positive for nuclear p65-NFĸB staining (nuclear labeling index, NLI) as a function of the parasite load under static conditions. Infected HbAA and HbAS erythrocytes (iHbAA and iHbAS) were analyzed. TNF-α served as a positive control and uninfected HbAA and HbAS red blood cells (HbAA and HbAS) were used as negative controls. As an additional control, the effect of an isogenic knobless FCR3 line (iHbAA K−) was investigated. The mean ± SEM of at least three independent biological replicates is shown, with at least 50 endothelial cells being analyzed per condition and per biological replicate. Note the data obtained for parasitized HbAA and HbAS as well as for the HbAA erythrocytes infected with the knobless parasite line are significantly different, as determined using F -tests ( F = 28; DF = 4; p
    Figure Legend Snippet: Reduced endothelial cell activation by infected HbAS erythrocytes. a Representative confocal microscopy images showing the subcellular localization of p65-NFĸB subunit in human dermal microvascular endothelial cells (HDMEC) upon TNF-α treatment (100 units ml −1 for 2 h) or upon co-culture with parasitized HbAA or HbAS erythrocytes (1 × 10 7 infected red blood cells at the trophozoite stage for 2 h) under static conditions. Medium served as a negative control. The p65-NFĸB subunit was localized using a specific polyclonal rabbit antiserum (dilution 1:200) and an Alexa-488 conjugated goat anti-rabbit IgG antiserum (1:400) as secondary antibody. The nuclei were stained using Hoechst 33342. Scale bar, 20 µm. b Percentage of HDMECs positive for nuclear p65-NFĸB staining (nuclear labeling index, NLI) as a function of the parasite load under static conditions. Infected HbAA and HbAS erythrocytes (iHbAA and iHbAS) were analyzed. TNF-α served as a positive control and uninfected HbAA and HbAS red blood cells (HbAA and HbAS) were used as negative controls. As an additional control, the effect of an isogenic knobless FCR3 line (iHbAA K−) was investigated. The mean ± SEM of at least three independent biological replicates is shown, with at least 50 endothelial cells being analyzed per condition and per biological replicate. Note the data obtained for parasitized HbAA and HbAS as well as for the HbAA erythrocytes infected with the knobless parasite line are significantly different, as determined using F -tests ( F = 28; DF = 4; p

    Techniques Used: Activation Assay, Infection, Confocal Microscopy, Co-Culture Assay, Negative Control, Staining, Labeling, Positive Control

    41) Product Images from "YB-1 regulates tumor growth by promoting MACC1/c-Met pathway in human lung adenocarcinoma"

    Article Title: YB-1 regulates tumor growth by promoting MACC1/c-Met pathway in human lung adenocarcinoma

    Journal: Oncotarget

    doi: 10.18632/oncotarget.18262

    YB-1 promotes MACC1 transcription by binding to MACC1 promoter and activates MACC1/c-Met pathway (A) Analysis of the MACC1 promoter indicated two putative YB-1 binding sites where the black boxes indicate sequences. RT-PCR analysis (B) and western blot analysis (C) determined the expression of YB-1 and MACC1 after inhibition of YB-1 in A549 cells. (D) MACC1 promoter (-2020 to +262) activity was analyzed by dual-luciferase reporter assay. The YB-1-silenced A549 cells and their corresponding control cells were co-transfected with plvx control plasmid or plvx-YB1 plasmid and MACC1 promoter (-2020 to +262) or basic reporter along with pRL-TK for 24h. (E) Mutated MACC1 promoter plasmids were generated (pGL3- MACC1 -mutated1; pGL3- MACC1 -mutated2). YB-1 binding sites in these two plasmids were mutated at two base pairs (pGL3- MACC1 -mutated1: -1860 to -1856; pGL3- MACC1 -mutated2: -1468 to -1464). A549 cells were transiently co-transfected with plvx vector plasmid or plvx-YB1 plasmid and the pGL3-basic reporter, the wild type MACC1 promoter reporter (pGL3- MACC1 ) or the mutated MACC1 promoter plasmids (pGL3-MACC1-mutated1; pGL3-MACC1-mutated2) along with pRL-TK for 24h. Transfected cells were harvested for dual-luciferase reporter assay. (F) ChIP assay was performed with YB-1 antibody or non-immune IgG as negative control. Immunoprecipitated DNA was amplified by PCR using primers as indicated. (G) Western blot analysis of YB-1, MACC1 and c-Met expression after inhibition of YB-1 in A549 cell. * P
    Figure Legend Snippet: YB-1 promotes MACC1 transcription by binding to MACC1 promoter and activates MACC1/c-Met pathway (A) Analysis of the MACC1 promoter indicated two putative YB-1 binding sites where the black boxes indicate sequences. RT-PCR analysis (B) and western blot analysis (C) determined the expression of YB-1 and MACC1 after inhibition of YB-1 in A549 cells. (D) MACC1 promoter (-2020 to +262) activity was analyzed by dual-luciferase reporter assay. The YB-1-silenced A549 cells and their corresponding control cells were co-transfected with plvx control plasmid or plvx-YB1 plasmid and MACC1 promoter (-2020 to +262) or basic reporter along with pRL-TK for 24h. (E) Mutated MACC1 promoter plasmids were generated (pGL3- MACC1 -mutated1; pGL3- MACC1 -mutated2). YB-1 binding sites in these two plasmids were mutated at two base pairs (pGL3- MACC1 -mutated1: -1860 to -1856; pGL3- MACC1 -mutated2: -1468 to -1464). A549 cells were transiently co-transfected with plvx vector plasmid or plvx-YB1 plasmid and the pGL3-basic reporter, the wild type MACC1 promoter reporter (pGL3- MACC1 ) or the mutated MACC1 promoter plasmids (pGL3-MACC1-mutated1; pGL3-MACC1-mutated2) along with pRL-TK for 24h. Transfected cells were harvested for dual-luciferase reporter assay. (F) ChIP assay was performed with YB-1 antibody or non-immune IgG as negative control. Immunoprecipitated DNA was amplified by PCR using primers as indicated. (G) Western blot analysis of YB-1, MACC1 and c-Met expression after inhibition of YB-1 in A549 cell. * P

    Techniques Used: Binding Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Inhibition, Activity Assay, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Generated, Chromatin Immunoprecipitation, Negative Control, Immunoprecipitation, Amplification, Polymerase Chain Reaction

    42) Product Images from "Duck enteritis virus UL21 is a late gene encoding a protein that interacts with pUL16"

    Article Title: Duck enteritis virus UL21 is a late gene encoding a protein that interacts with pUL16

    Journal: BMC Veterinary Research

    doi: 10.1186/s12917-019-2228-7

    Localization of DEV UL21 in infected and transfected DEFs. A: DEV-infected cells on coverslips were fixed. The samples were incubated successively with rabbit anti-UL21 IgG and goat anti-rabbit IgG conjugated with Alexa Fluor 594. We captured the images by fluorescence microscopy using a 40× objective. B: the DEFs were transfected with pUL21 to observe localization. The samples were incubated successively with rabbit anti-UL21 IgG and goat anti-rabbit IgG conjugated with Alexa Fluor 488. The images were captured under fluorescence microscopy using a 40× objective
    Figure Legend Snippet: Localization of DEV UL21 in infected and transfected DEFs. A: DEV-infected cells on coverslips were fixed. The samples were incubated successively with rabbit anti-UL21 IgG and goat anti-rabbit IgG conjugated with Alexa Fluor 594. We captured the images by fluorescence microscopy using a 40× objective. B: the DEFs were transfected with pUL21 to observe localization. The samples were incubated successively with rabbit anti-UL21 IgG and goat anti-rabbit IgG conjugated with Alexa Fluor 488. The images were captured under fluorescence microscopy using a 40× objective

    Techniques Used: Infection, Transfection, Incubation, Fluorescence, Microscopy

    43) Product Images from "Procoagulant and immunogenic properties of melanoma exosomes, microvesicles and apoptotic vesicles"

    Article Title: Procoagulant and immunogenic properties of melanoma exosomes, microvesicles and apoptotic vesicles

    Journal: Oncotarget

    doi: 10.18632/oncotarget.10783

    Surface expression comparison of B16-F1-derived vesicles using flow cytometry ( A ) Vesicles conjugated to aldehyde-sulfate microspheres were analyzed by flow cytometry using biotinylated (bio) antibodies for the indicated tetraspanins, adhesion molecules, and clotting factors; bio-annexin V for phosphatidylserine (PS) and goat anti-mouse tissue factor (TF). ( B ) TF expression on B16-F1 cells, EL4, or TF-transfected EL4 (EL4-TF) as analyzed by flow cytometry. Soluble TF (sTF) was used to neutralise the anti-TF polyclonal antibody. Biotin was detected using streptavidin-allophycocyanin (SA-APC), and TF was detected using rabbit anti-goat IgG Alexa Fluor ® 594. Grey shaded peaks represent BSA-bead control, goat IgG control for TF bead samples; black lines represent EV-beads or cells; dotted lines represent TF antibody neutralized cells. ( C ) Vesicle lysates were subjected to PAGE and Western blotted with goat anti-mouse CD147 (detected with anti-goat IgG-horse radish peroxidase (HRP), mouse anti-mouse clathrin heavy chain, and mouse anti-mouse β actin IgG-HRP (detected with anti-mouse IgG-HRP). MW in kDa are shown. Results are representative of at least two experiments. ( D ) TOP3 precursor ion intensities [ 69 ] normalised to β-actin (y-axis) are represented in rank order (x-axis) in the exosome proteome for the three vesicle types.
    Figure Legend Snippet: Surface expression comparison of B16-F1-derived vesicles using flow cytometry ( A ) Vesicles conjugated to aldehyde-sulfate microspheres were analyzed by flow cytometry using biotinylated (bio) antibodies for the indicated tetraspanins, adhesion molecules, and clotting factors; bio-annexin V for phosphatidylserine (PS) and goat anti-mouse tissue factor (TF). ( B ) TF expression on B16-F1 cells, EL4, or TF-transfected EL4 (EL4-TF) as analyzed by flow cytometry. Soluble TF (sTF) was used to neutralise the anti-TF polyclonal antibody. Biotin was detected using streptavidin-allophycocyanin (SA-APC), and TF was detected using rabbit anti-goat IgG Alexa Fluor ® 594. Grey shaded peaks represent BSA-bead control, goat IgG control for TF bead samples; black lines represent EV-beads or cells; dotted lines represent TF antibody neutralized cells. ( C ) Vesicle lysates were subjected to PAGE and Western blotted with goat anti-mouse CD147 (detected with anti-goat IgG-horse radish peroxidase (HRP), mouse anti-mouse clathrin heavy chain, and mouse anti-mouse β actin IgG-HRP (detected with anti-mouse IgG-HRP). MW in kDa are shown. Results are representative of at least two experiments. ( D ) TOP3 precursor ion intensities [ 69 ] normalised to β-actin (y-axis) are represented in rank order (x-axis) in the exosome proteome for the three vesicle types.

    Techniques Used: Expressing, Derivative Assay, Flow Cytometry, Cytometry, Coagulation, Transfection, Polyacrylamide Gel Electrophoresis, Western Blot

    44) Product Images from "Peptide‐based tumor inhibitor encoding mitochondrial p14ARF is highly efficacious to diverse tumors"

    Article Title: Peptide‐based tumor inhibitor encoding mitochondrial p14ARF is highly efficacious to diverse tumors

    Journal: Cancer Science

    doi: 10.1111/cas.12991

    Intracellular localization of endogenous p14 ARF in tumor cells of diverse origins and non‐neoplastic cells. (a) Double immunofluorescence using both anti‐p14 ARF (1:500 dilution) and anti‐HSP60 (1:2000 dil.) antibodies for various lineages of human tumor cells. (b) Immunoelectron microscopy for HeLa cells using rabbit anti‐p14 polyAb (1:100 dil.) and 10 nm gold particle‐conjugated goat anti‐rabbit IgG polyAb. (c) Double immunofluorescence for non‐neoplastic cells of normal origins expressing p14 and HSP60. (d) Expression of endogenous p14 ARF both in the tumor cells and normal lineage cells by RT‐PCR. β‐actin was amplified as an endogenous control to p14 mRNA.
    Figure Legend Snippet: Intracellular localization of endogenous p14 ARF in tumor cells of diverse origins and non‐neoplastic cells. (a) Double immunofluorescence using both anti‐p14 ARF (1:500 dilution) and anti‐HSP60 (1:2000 dil.) antibodies for various lineages of human tumor cells. (b) Immunoelectron microscopy for HeLa cells using rabbit anti‐p14 polyAb (1:100 dil.) and 10 nm gold particle‐conjugated goat anti‐rabbit IgG polyAb. (c) Double immunofluorescence for non‐neoplastic cells of normal origins expressing p14 and HSP60. (d) Expression of endogenous p14 ARF both in the tumor cells and normal lineage cells by RT‐PCR. β‐actin was amplified as an endogenous control to p14 mRNA.

    Techniques Used: Immunofluorescence, Immuno-Electron Microscopy, Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification

    45) Product Images from "Primary rat LSECs preserve their characteristic phenotype after cryopreservation"

    Article Title: Primary rat LSECs preserve their characteristic phenotype after cryopreservation

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-32103-z

    Expression of main endocytosis receptors by fLSEC and cLSECs. In all micrographs, the fLSECs are shown in the left panel and cLSECs in the right panel. The cultures were fixed with paraformaldehyde, permeabilized with Triton X, and immune labeled with antibodies against stabilin-2 ( a and b ), mannose receptor (MR) ( d and e ), and FcγRIIb2 ( g and h ). Positive immunolabeling was visualized with Alexa Fluor-488 secondary antibodies (green fluorescence). Cell nuclei were stained with DAPI (blue fluorescence). ( g — i ) The average fluorescence intensity per cell for each receptor protein was measured and the results expressed as relative expression, where the expression of the different markers in fLSECs equals 1. The p value is shown, which was calculated using the Excel two-tailed paired t -test assuming unequal variation. Statistical details are presented in Table 2 under Materials and Methods.
    Figure Legend Snippet: Expression of main endocytosis receptors by fLSEC and cLSECs. In all micrographs, the fLSECs are shown in the left panel and cLSECs in the right panel. The cultures were fixed with paraformaldehyde, permeabilized with Triton X, and immune labeled with antibodies against stabilin-2 ( a and b ), mannose receptor (MR) ( d and e ), and FcγRIIb2 ( g and h ). Positive immunolabeling was visualized with Alexa Fluor-488 secondary antibodies (green fluorescence). Cell nuclei were stained with DAPI (blue fluorescence). ( g — i ) The average fluorescence intensity per cell for each receptor protein was measured and the results expressed as relative expression, where the expression of the different markers in fLSECs equals 1. The p value is shown, which was calculated using the Excel two-tailed paired t -test assuming unequal variation. Statistical details are presented in Table 2 under Materials and Methods.

    Techniques Used: Expressing, Labeling, Immunolabeling, Fluorescence, Staining, Two Tailed Test

    46) Product Images from "Alga-Produced Cholera Toxin-Pfs25 Fusion Proteins as Oral Vaccines"

    Article Title: Alga-Produced Cholera Toxin-Pfs25 Fusion Proteins as Oral Vaccines

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00714-13

    Immunogenicity of alga-produced CtxB-Pfs25 and Pfs5 using Alhydrogel. Mice were vaccinated by intraperitoneal injection with 20 μg of affinity-purified Cr.CtxB-Pfs25 or the molar equivalent of Cr.Pfs25 adsorbed to Alhydrogel. Pfs25-specific IgG
    Figure Legend Snippet: Immunogenicity of alga-produced CtxB-Pfs25 and Pfs5 using Alhydrogel. Mice were vaccinated by intraperitoneal injection with 20 μg of affinity-purified Cr.CtxB-Pfs25 or the molar equivalent of Cr.Pfs25 adsorbed to Alhydrogel. Pfs25-specific IgG

    Techniques Used: Produced, Mouse Assay, Injection, Affinity Purification

    Immune response to whole-cell alga oral immunization in naive mice. Mice were administered weekly doses of JAG101 algae or Δ psbA C. reinhardtii . Serum IgG antibodies for Pfs25 (A) and CtxB (B) were detected by ELISA. Error bars indicate standard
    Figure Legend Snippet: Immune response to whole-cell alga oral immunization in naive mice. Mice were administered weekly doses of JAG101 algae or Δ psbA C. reinhardtii . Serum IgG antibodies for Pfs25 (A) and CtxB (B) were detected by ELISA. Error bars indicate standard

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    47) Product Images from "Conserved Active-Site Residues Associated with OAS Enzyme Activity and Ubiquitin-Like Domains Are Not Required for the Antiviral Activity of goOASL Protein against Avian Tembusu Virus"

    Article Title: Conserved Active-Site Residues Associated with OAS Enzyme Activity and Ubiquitin-Like Domains Are Not Required for the Antiviral Activity of goOASL Protein against Avian Tembusu Virus

    Journal: Viruses

    doi: 10.3390/v10070371

    Cellular localization of goOASL and its mutant proteins in DEFs. ( A ) Cellular localization of goOASL and its mutant proteins in normal DEFs. DEFs seeded on 20-mm glass slides in a 12-well tissue culture plate were transfected with goOASL, goOASL-S64C/D76E/D78E/D144T, goOASL∆UBLs, or goOASL∆UBLs-S64C/D76E/D78E/D144T (1.6 µg/well). Immunofluorescence was detected at 36 h after transfection via fluorescence microscopy; ( B ) Cellular colocalization of goOASL and its mutant proteins in DEFs with DTMUV. DEFs seeded on the 20-mm glass slides in the 12-well tissue culture plate were transfected with goOASL, goOASL-S64C/D76E/D78E/D144T, goOASL∆UBLs, or goOASL∆UBLs-S64C/D76E/D78E/D144T (1.6 µg/well). At 24 h after transfection, the cells were infected with DTMUV (10 4 TCID 50 /well) for another 12 h. Immunofluorescence was detected using fluorescence microscopy. The rabbit anti-His antibody and mouse anti-DTMUV antibody were used as primary antibodies, the TRITC-goat anti-rabbit IgG and FITC-goat anti-mouse IgG were used as secondary antibodies. DAPI was used for nucleolus staining. Fluorescence (red, green and blue) was detected via fluorescence microscopy (magnification 600×) and analysed using Image Pro Plus 6.0.
    Figure Legend Snippet: Cellular localization of goOASL and its mutant proteins in DEFs. ( A ) Cellular localization of goOASL and its mutant proteins in normal DEFs. DEFs seeded on 20-mm glass slides in a 12-well tissue culture plate were transfected with goOASL, goOASL-S64C/D76E/D78E/D144T, goOASL∆UBLs, or goOASL∆UBLs-S64C/D76E/D78E/D144T (1.6 µg/well). Immunofluorescence was detected at 36 h after transfection via fluorescence microscopy; ( B ) Cellular colocalization of goOASL and its mutant proteins in DEFs with DTMUV. DEFs seeded on the 20-mm glass slides in the 12-well tissue culture plate were transfected with goOASL, goOASL-S64C/D76E/D78E/D144T, goOASL∆UBLs, or goOASL∆UBLs-S64C/D76E/D78E/D144T (1.6 µg/well). At 24 h after transfection, the cells were infected with DTMUV (10 4 TCID 50 /well) for another 12 h. Immunofluorescence was detected using fluorescence microscopy. The rabbit anti-His antibody and mouse anti-DTMUV antibody were used as primary antibodies, the TRITC-goat anti-rabbit IgG and FITC-goat anti-mouse IgG were used as secondary antibodies. DAPI was used for nucleolus staining. Fluorescence (red, green and blue) was detected via fluorescence microscopy (magnification 600×) and analysed using Image Pro Plus 6.0.

    Techniques Used: Mutagenesis, Transfection, Immunofluorescence, Fluorescence, Microscopy, Infection, Staining

    48) Product Images from "Conserved Active-Site Residues Associated with OAS Enzyme Activity and Ubiquitin-Like Domains Are Not Required for the Antiviral Activity of goOASL Protein against Avian Tembusu Virus"

    Article Title: Conserved Active-Site Residues Associated with OAS Enzyme Activity and Ubiquitin-Like Domains Are Not Required for the Antiviral Activity of goOASL Protein against Avian Tembusu Virus

    Journal: Viruses

    doi: 10.3390/v10070371

    Cellular localization of goOASL and its mutant proteins in DEFs. ( A ) Cellular localization of goOASL and its mutant proteins in normal DEFs. DEFs seeded on 20-mm glass slides in a 12-well tissue culture plate were transfected with goOASL, goOASL-S64C/D76E/D78E/D144T, goOASL∆UBLs, or goOASL∆UBLs-S64C/D76E/D78E/D144T (1.6 µg/well). Immunofluorescence was detected at 36 h after transfection via fluorescence microscopy; ( B ) Cellular colocalization of goOASL and its mutant proteins in DEFs with DTMUV. DEFs seeded on the 20-mm glass slides in the 12-well tissue culture plate were transfected with goOASL, goOASL-S64C/D76E/D78E/D144T, goOASL∆UBLs, or goOASL∆UBLs-S64C/D76E/D78E/D144T (1.6 µg/well). At 24 h after transfection, the cells were infected with DTMUV (10 4 TCID 50 /well) for another 12 h. Immunofluorescence was detected using fluorescence microscopy. The rabbit anti-His antibody and mouse anti-DTMUV antibody were used as primary antibodies, the TRITC-goat anti-rabbit IgG and FITC-goat anti-mouse IgG were used as secondary antibodies. DAPI was used for nucleolus staining. Fluorescence (red, green and blue) was detected via fluorescence microscopy (magnification 600×) and analysed using Image Pro Plus 6.0.
    Figure Legend Snippet: Cellular localization of goOASL and its mutant proteins in DEFs. ( A ) Cellular localization of goOASL and its mutant proteins in normal DEFs. DEFs seeded on 20-mm glass slides in a 12-well tissue culture plate were transfected with goOASL, goOASL-S64C/D76E/D78E/D144T, goOASL∆UBLs, or goOASL∆UBLs-S64C/D76E/D78E/D144T (1.6 µg/well). Immunofluorescence was detected at 36 h after transfection via fluorescence microscopy; ( B ) Cellular colocalization of goOASL and its mutant proteins in DEFs with DTMUV. DEFs seeded on the 20-mm glass slides in the 12-well tissue culture plate were transfected with goOASL, goOASL-S64C/D76E/D78E/D144T, goOASL∆UBLs, or goOASL∆UBLs-S64C/D76E/D78E/D144T (1.6 µg/well). At 24 h after transfection, the cells were infected with DTMUV (10 4 TCID 50 /well) for another 12 h. Immunofluorescence was detected using fluorescence microscopy. The rabbit anti-His antibody and mouse anti-DTMUV antibody were used as primary antibodies, the TRITC-goat anti-rabbit IgG and FITC-goat anti-mouse IgG were used as secondary antibodies. DAPI was used for nucleolus staining. Fluorescence (red, green and blue) was detected via fluorescence microscopy (magnification 600×) and analysed using Image Pro Plus 6.0.

    Techniques Used: Mutagenesis, Transfection, Immunofluorescence, Fluorescence, Microscopy, Infection, Staining

    49) Product Images from "Role of SMC1 in Overcoming Drug Resistance in Triple Negative Breast Cancer"

    Article Title: Role of SMC1 in Overcoming Drug Resistance in Triple Negative Breast Cancer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0064338

    Cellular localization of SMC1 in MDA-MB-231 Cells. Immunocytological localization SMC1 was performed on MDA-MB-231 fixed cells by method described previously with slight modifications [37] , [38] , [40] , [41] . Cells were grown on glass cover slips and fixed with ice-cold methanol and acetic acid (3∶1). Nonspecific antibody interactions were minimized by pre-treating the cells with 10% goat serum in PBS for 60 min at room temperature. The cells were subjected to immuno-cytochemistry using anti-SMC1 IgG (raised in rabbit) as a primary antibody and goat-anti-rabbit Rhodamine red-x-conjugated secondary antibody. DAPI (4′, 6-Diamidino-2-phenylindole) was used as a nuclear counter-stain. Slides were analyzed by confocal laser microscope (Zeiss LSM510 META, Germany) at 40× magnification ( Panel A ). Surface localization of SMC1 was determined by flow cytometry using indirect flow cytometry protocol [40] . Briefly, MDA-MB-231 cells were harvested and resuspended to approximately 1×10 6 cell/ml in ice-cold PBS, 10% FBA and 1% sodium azide. Cells were incubated with 1 µg/ml anti-SMC1 IgG in 3% BSA/PBS solution and incubated at 4°C for 2 hour followed by washing with PBS and incubated with FITC-conjugated secondary antibody for 30 min at room temperature in dark. Cells were washed with PBS 3–5 times and resuspended in ice-cold PBS containing 3% BSA and 1% sodium azide and analyze by flow-cytometry ( Panel B ). Subcellular distribution of SMC1 was determined in the MDA-MB-231 cells using subcellular protein fractionation kit (Thermo Scientific) as detailed in methods section. Immuno-precipitation was performed in all the 3 fractions using anti-SMC1 IgG using the protocol as described in method section. All the immune-precipitates were characterized by western blot using anti-SMC1 and anti-SMC3 IgG ( Panel C ).
    Figure Legend Snippet: Cellular localization of SMC1 in MDA-MB-231 Cells. Immunocytological localization SMC1 was performed on MDA-MB-231 fixed cells by method described previously with slight modifications [37] , [38] , [40] , [41] . Cells were grown on glass cover slips and fixed with ice-cold methanol and acetic acid (3∶1). Nonspecific antibody interactions were minimized by pre-treating the cells with 10% goat serum in PBS for 60 min at room temperature. The cells were subjected to immuno-cytochemistry using anti-SMC1 IgG (raised in rabbit) as a primary antibody and goat-anti-rabbit Rhodamine red-x-conjugated secondary antibody. DAPI (4′, 6-Diamidino-2-phenylindole) was used as a nuclear counter-stain. Slides were analyzed by confocal laser microscope (Zeiss LSM510 META, Germany) at 40× magnification ( Panel A ). Surface localization of SMC1 was determined by flow cytometry using indirect flow cytometry protocol [40] . Briefly, MDA-MB-231 cells were harvested and resuspended to approximately 1×10 6 cell/ml in ice-cold PBS, 10% FBA and 1% sodium azide. Cells were incubated with 1 µg/ml anti-SMC1 IgG in 3% BSA/PBS solution and incubated at 4°C for 2 hour followed by washing with PBS and incubated with FITC-conjugated secondary antibody for 30 min at room temperature in dark. Cells were washed with PBS 3–5 times and resuspended in ice-cold PBS containing 3% BSA and 1% sodium azide and analyze by flow-cytometry ( Panel B ). Subcellular distribution of SMC1 was determined in the MDA-MB-231 cells using subcellular protein fractionation kit (Thermo Scientific) as detailed in methods section. Immuno-precipitation was performed in all the 3 fractions using anti-SMC1 IgG using the protocol as described in method section. All the immune-precipitates were characterized by western blot using anti-SMC1 and anti-SMC3 IgG ( Panel C ).

    Techniques Used: Multiple Displacement Amplification, Immunocytochemistry, Staining, Microscopy, Flow Cytometry, Cytometry, Incubation, Fractionation, Immunoprecipitation, Western Blot

    50) Product Images from "Expression of area-specific M2-macrophage phenotype by recruited rat monocytes in duct-ligation pancreatitis"

    Article Title: Expression of area-specific M2-macrophage phenotype by recruited rat monocytes in duct-ligation pancreatitis

    Journal: Histochemistry and Cell Biology

    doi: 10.1007/s00418-016-1406-y

    Expression of CD206 by macrophages with different phenotypes in the interacinar area of Lewis rat pancreas on day 4 after duct-ligation surgery. Three-color immunofluorescence staining for CD206 ( d , f Alexa-594-conjugated anti-rabbit IgG, blue ), CD68 ( a , indirect staining with Alexa-488-conjugated anti-mouse IgG, green ), and CD163 ( b , indirect staining with AMCA-conjugated streptavidin, red ). c and e Merged images. A differential interference contrast image is superimposed onto each picture except for c . Note that CD68 + CD163 + CD206 + cells are mostly confined to the interlobular area ( L ) and very few are in the interacinar area ( A ). Pseudocolors are assigned using AxioVision software. Scale bars c 100 μm; e 20 μm. g Number of each phenotype/mm 2 of the interacinar area (mean ± SD, n = 3 rats each). Note that there are only a few CD206 + and CD163 + cells, indicating that CD68 + cells upregulated neither CD206 nor CD163
    Figure Legend Snippet: Expression of CD206 by macrophages with different phenotypes in the interacinar area of Lewis rat pancreas on day 4 after duct-ligation surgery. Three-color immunofluorescence staining for CD206 ( d , f Alexa-594-conjugated anti-rabbit IgG, blue ), CD68 ( a , indirect staining with Alexa-488-conjugated anti-mouse IgG, green ), and CD163 ( b , indirect staining with AMCA-conjugated streptavidin, red ). c and e Merged images. A differential interference contrast image is superimposed onto each picture except for c . Note that CD68 + CD163 + CD206 + cells are mostly confined to the interlobular area ( L ) and very few are in the interacinar area ( A ). Pseudocolors are assigned using AxioVision software. Scale bars c 100 μm; e 20 μm. g Number of each phenotype/mm 2 of the interacinar area (mean ± SD, n = 3 rats each). Note that there are only a few CD206 + and CD163 + cells, indicating that CD68 + cells upregulated neither CD206 nor CD163

    Techniques Used: Expressing, Ligation, Immunofluorescence, Staining, Software

    Expression of arginase 1 ( a – f ) by macrophages with different phenotypes in the interlobular area and that of NOS2 ( g – l ) by macrophages in the interacinar area of Lewis rat pancreas on day 4 after duct-ligation surgery. a and g Three-color immunoenzyme staining for arginase 1 ( a , blue ) or NOS2 ( g , blue ), type IV collagen-like molecules (B12, brown ), and BrdU ( red ). Note preferential localization of arginase 1 + cells in the interlobular area ( L ) but not in the interacinar area ( A ). In contrast, NOS2 + cells are mainly in the interacinar area ( A ). b – e and h – k Three-color immunofluorescence staining for arginase 1 or NOS2 ( d or j , Alexa-594-conjugated anti-rabbit IgG, blue ), CD68 ( b and h , indirect staining with Alexa-488-conjugated anti-mouse IgG, green ), and CD163 ( c and i , indirect staining with AMCA-conjugated streptavidin, red ). e and k Merged images. Differential interference contrast image is superimposed onto each picture. Arrows indicate arginase 1 + CD68 + CD163 + , black arrowhead is arginase 1 + CD68 + CD163 − , and white arrowhead is arginase 1 − CD68 + CD163 + cells. Pseudocolors are assigned using AxioVision software. Scale bars c 100 μm; e 20 μm. f and l The proportion of each phenotype in the lobular (f) or interacinar areas (l) (mean ± SD, n = 3 rats each). Note that most of CD68 + CD163 + cells and ~60 % of CD68 + CD163 − cells are arginase 1 + on day 4 in the interlobular area. In contrast, in the interacinar area, CD68 + CD163 − cells partly expressed NOS2
    Figure Legend Snippet: Expression of arginase 1 ( a – f ) by macrophages with different phenotypes in the interlobular area and that of NOS2 ( g – l ) by macrophages in the interacinar area of Lewis rat pancreas on day 4 after duct-ligation surgery. a and g Three-color immunoenzyme staining for arginase 1 ( a , blue ) or NOS2 ( g , blue ), type IV collagen-like molecules (B12, brown ), and BrdU ( red ). Note preferential localization of arginase 1 + cells in the interlobular area ( L ) but not in the interacinar area ( A ). In contrast, NOS2 + cells are mainly in the interacinar area ( A ). b – e and h – k Three-color immunofluorescence staining for arginase 1 or NOS2 ( d or j , Alexa-594-conjugated anti-rabbit IgG, blue ), CD68 ( b and h , indirect staining with Alexa-488-conjugated anti-mouse IgG, green ), and CD163 ( c and i , indirect staining with AMCA-conjugated streptavidin, red ). e and k Merged images. Differential interference contrast image is superimposed onto each picture. Arrows indicate arginase 1 + CD68 + CD163 + , black arrowhead is arginase 1 + CD68 + CD163 − , and white arrowhead is arginase 1 − CD68 + CD163 + cells. Pseudocolors are assigned using AxioVision software. Scale bars c 100 μm; e 20 μm. f and l The proportion of each phenotype in the lobular (f) or interacinar areas (l) (mean ± SD, n = 3 rats each). Note that most of CD68 + CD163 + cells and ~60 % of CD68 + CD163 − cells are arginase 1 + on day 4 in the interlobular area. In contrast, in the interacinar area, CD68 + CD163 − cells partly expressed NOS2

    Techniques Used: Expressing, Ligation, Staining, Immunofluorescence, Software

    Expression of CD206 by macrophages with different phenotypes in the interlobular area of Lewis rat pancreas on day 4 after duct-ligation surgery. a – d Three-color immunofluorescence staining for CD206 ( c , Alexa-594-conjugated anti-rabbit IgG, blue ), CD68 ( a , indirect staining with Alexa-488-conjugated anti-mouse IgG, green ), and CD163 ( b , indirect staining with AMCA-conjugated streptavidin, red ). d Merged image of a – c . Arrows indicate CD68 + CD163 high CD206 + or CD68 + CD163 low CD206 + cells. Black arrowhead indicates CD68 + CD163 − CD206 + cell and white arrowhead CD68 + CD163 − CD206 − cell. Pseudocolors are assigned using AxioVision software. Scale bar d 10 μm. e Proportions of each phenotype in the interlobular area (mean ± SD, n = 3 rats each). Both CD68 + CD163 low CD206 + cells and CD68 + CD163 high CD206 + cells significantly increased in number from day 1 to day 2 (* p
    Figure Legend Snippet: Expression of CD206 by macrophages with different phenotypes in the interlobular area of Lewis rat pancreas on day 4 after duct-ligation surgery. a – d Three-color immunofluorescence staining for CD206 ( c , Alexa-594-conjugated anti-rabbit IgG, blue ), CD68 ( a , indirect staining with Alexa-488-conjugated anti-mouse IgG, green ), and CD163 ( b , indirect staining with AMCA-conjugated streptavidin, red ). d Merged image of a – c . Arrows indicate CD68 + CD163 high CD206 + or CD68 + CD163 low CD206 + cells. Black arrowhead indicates CD68 + CD163 − CD206 + cell and white arrowhead CD68 + CD163 − CD206 − cell. Pseudocolors are assigned using AxioVision software. Scale bar d 10 μm. e Proportions of each phenotype in the interlobular area (mean ± SD, n = 3 rats each). Both CD68 + CD163 low CD206 + cells and CD68 + CD163 high CD206 + cells significantly increased in number from day 1 to day 2 (* p

    Techniques Used: Expressing, Ligation, Immunofluorescence, Staining, Software

    Infiltration of GFP + monocytes in the GFP + leukocyte-transfer experiment. a – e Three-color immunoenzyme staining for GFP ( c – e , blue ) or CD68 ( a , blue ), type IV collagen ( brown ), and BrdU ( red ). Day 2 ( a and d) and day 4 ( b and e ) after duct-ligation surgery and sham-operated control ( c ). b Anti-GFP antibody is omitted (Ab−) showing no background GFP staining. Note that GFP + cells increased in number over time but are fewer compared to CD68 + cells on day 2 ( d vs. a ). A few GFP + cells are BrdU + ( inset of d ). f – j Four-color immunofluorescence staining for CD68 ( g , indirect staining with Alexa-594-conjugated anti-mouse IgG, green ), GFP ( f , Alexa-488–conjugated anti-GFP, green ), CD163 ( h , biotin-labeled mAb plus AMCA-conjugated streptavidin, red ), EdU ( i , Alexa-647–conjugated azide, purple ). j Merged image of f – i . Arrows indicate either GFP + CD68 + CD163 high EdU + or GFP + CD68 + CD163 low EdU + cells. Pseudocolors are assigned using AxioVision software. Scale bars : a 200 μm; b – e 100 μm; inset of d 20 μm; j 10 μm
    Figure Legend Snippet: Infiltration of GFP + monocytes in the GFP + leukocyte-transfer experiment. a – e Three-color immunoenzyme staining for GFP ( c – e , blue ) or CD68 ( a , blue ), type IV collagen ( brown ), and BrdU ( red ). Day 2 ( a and d) and day 4 ( b and e ) after duct-ligation surgery and sham-operated control ( c ). b Anti-GFP antibody is omitted (Ab−) showing no background GFP staining. Note that GFP + cells increased in number over time but are fewer compared to CD68 + cells on day 2 ( d vs. a ). A few GFP + cells are BrdU + ( inset of d ). f – j Four-color immunofluorescence staining for CD68 ( g , indirect staining with Alexa-594-conjugated anti-mouse IgG, green ), GFP ( f , Alexa-488–conjugated anti-GFP, green ), CD163 ( h , biotin-labeled mAb plus AMCA-conjugated streptavidin, red ), EdU ( i , Alexa-647–conjugated azide, purple ). j Merged image of f – i . Arrows indicate either GFP + CD68 + CD163 high EdU + or GFP + CD68 + CD163 low EdU + cells. Pseudocolors are assigned using AxioVision software. Scale bars : a 200 μm; b – e 100 μm; inset of d 20 μm; j 10 μm

    Techniques Used: Staining, Ligation, Immunofluorescence, Labeling, Software

    Expression of CD206 by GFP + monocytes in the GFP + leukocyte-transfer experiment. Day 2 ( a , b , d , e ) and day 4 ( f – i) after duct-ligation surgery and sham-operated control ( c ). a – e Two-color immunoenzyme staining for CD206 ( c – e , blue ) or CD163 ( a , blue ) and BrdU ( red ). b Anti-CD206 antibody is omitted (Ab−), showing no background CD206 staining. Note that the number of CD163 + cells ( a ) is comparable to that of CD206 + cells ( d ) and some CD206 + cells are BrdU + ( e , rectangular area of d ). f – i Three-color immunofluorescence staining for CD206 ( g , Alexa-594-conjugated anti-rabbit IgG, blue ), GFP ( e , Alexa-488-conjugated anti-GFP, green ), CD163 (biotin-labeled mAb plus AMCA-conjugated streptavidin, red ). i Merged image of ( f – h ). Arrows indicate either GFP + CD68 + CD163 high or GFP + CD68 + CD163 low cells. Pseudocolors are assigned using AxioVision software. Scale bars a 200 μm; b – d 100 μm; e 20 μm; i 10 μm
    Figure Legend Snippet: Expression of CD206 by GFP + monocytes in the GFP + leukocyte-transfer experiment. Day 2 ( a , b , d , e ) and day 4 ( f – i) after duct-ligation surgery and sham-operated control ( c ). a – e Two-color immunoenzyme staining for CD206 ( c – e , blue ) or CD163 ( a , blue ) and BrdU ( red ). b Anti-CD206 antibody is omitted (Ab−), showing no background CD206 staining. Note that the number of CD163 + cells ( a ) is comparable to that of CD206 + cells ( d ) and some CD206 + cells are BrdU + ( e , rectangular area of d ). f – i Three-color immunofluorescence staining for CD206 ( g , Alexa-594-conjugated anti-rabbit IgG, blue ), GFP ( e , Alexa-488-conjugated anti-GFP, green ), CD163 (biotin-labeled mAb plus AMCA-conjugated streptavidin, red ). i Merged image of ( f – h ). Arrows indicate either GFP + CD68 + CD163 high or GFP + CD68 + CD163 low cells. Pseudocolors are assigned using AxioVision software. Scale bars a 200 μm; b – d 100 μm; e 20 μm; i 10 μm

    Techniques Used: Expressing, Ligation, Staining, Immunofluorescence, Labeling, Software

    Four-color immunofluorescence staining for CD68 (indirect staining with Alexa-594-conjugated anti-mouse IgG, green ), CD163 (biotin-labeled mAb plus Alexa-488-conjugated streptavidin, red ), type IV collagen (indirect staining with AMCA-conjugated anti-rabbit IgG, blue ), and EdU (Alexa-647-conjugated azide, purple ). The interlobular area ( L ) and interacinar area ( A ) of Lewis rat pancreas on day 2 after duct-ligation surgery. The same place a – c and d – g is photomicrographed for CD68 plus type IV collagen ( a , d ), CD163 plus type IV collagen ( b , e ), EdU plus type IV collagen ( f ), and merged images ( c , g ), respectively. d – g Four different phenotypes of macrophages in the interlobular area: CD68 + CD163 − ( white arrowhead ), CD68 + CD163 low ( black arrowheads ), CD68 + CD163 high (also EdU + , arrow ), and CD68 − CD163 + (also EdU + , double arrows ) are clearly depicted. Pseudocolors are assigned using AxioVision software. Scale bars c 100 μm; g 20 μm
    Figure Legend Snippet: Four-color immunofluorescence staining for CD68 (indirect staining with Alexa-594-conjugated anti-mouse IgG, green ), CD163 (biotin-labeled mAb plus Alexa-488-conjugated streptavidin, red ), type IV collagen (indirect staining with AMCA-conjugated anti-rabbit IgG, blue ), and EdU (Alexa-647-conjugated azide, purple ). The interlobular area ( L ) and interacinar area ( A ) of Lewis rat pancreas on day 2 after duct-ligation surgery. The same place a – c and d – g is photomicrographed for CD68 plus type IV collagen ( a , d ), CD163 plus type IV collagen ( b , e ), EdU plus type IV collagen ( f ), and merged images ( c , g ), respectively. d – g Four different phenotypes of macrophages in the interlobular area: CD68 + CD163 − ( white arrowhead ), CD68 + CD163 low ( black arrowheads ), CD68 + CD163 high (also EdU + , arrow ), and CD68 − CD163 + (also EdU + , double arrows ) are clearly depicted. Pseudocolors are assigned using AxioVision software. Scale bars c 100 μm; g 20 μm

    Techniques Used: Immunofluorescence, Staining, Labeling, Ligation, Software

    51) Product Images from "Catalytically inactive receptor tyrosine kinase PTK7 activates FGFR1 independent of FGF"

    Article Title: Catalytically inactive receptor tyrosine kinase PTK7 activates FGFR1 independent of FGF

    Journal: The FASEB Journal

    doi: 10.1096/fj.201900932R

    Colocalization of PTK7 and FGFR1 in HEK293 cells. Serum-depleted HEK293 cells transiently expressing PTK7-FLAG and FGFR1-HA were fixed, permeabilized, and stained with mouse anti-FLAG, rabbit anti-HA antibodies, Alexa Fluor Rhodamine Red-conjugated goat anti-mouse IgG, Alexa Fluor 488–conjugated goat anti-rabbit IgG antibodies, and DAPI. The fluorescence signals were analyzed by confocal fluorescence microscopy. The confocal images on the upper and lower panels were taken at the bottom and in the middle of the cells, respectively. Scale bars, 10 μm.
    Figure Legend Snippet: Colocalization of PTK7 and FGFR1 in HEK293 cells. Serum-depleted HEK293 cells transiently expressing PTK7-FLAG and FGFR1-HA were fixed, permeabilized, and stained with mouse anti-FLAG, rabbit anti-HA antibodies, Alexa Fluor Rhodamine Red-conjugated goat anti-mouse IgG, Alexa Fluor 488–conjugated goat anti-rabbit IgG antibodies, and DAPI. The fluorescence signals were analyzed by confocal fluorescence microscopy. The confocal images on the upper and lower panels were taken at the bottom and in the middle of the cells, respectively. Scale bars, 10 μm.

    Techniques Used: Expressing, Staining, Fluorescence, Microscopy

    52) Product Images from "Expression and regulation of avian beta-defensin 8 protein in immune tissues and cell lines of chickens"

    Article Title: Expression and regulation of avian beta-defensin 8 protein in immune tissues and cell lines of chickens

    Journal: Asian-Australasian Journal of Animal Sciences

    doi: 10.5713/ajas.17.0836

    Determination of AvBD8 protein expression in immune cell lines by performing immunocytochemical analysis. Cultured chicken CU91 T-, DT40 B-, HD11 macrophage-, and OU2 fibroblast-cell lines were incubated with the rabbit anti-AvBD8 primary antibody, followed by incubation with the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody, and were counterstained with 4′,6-diamidino-2-phenylindole. Control cells were incubated with secondary antibody only. AvBD8, avian beta-defensin 8; IgG, immunoglobulin G. Scale bar: 20 μm.
    Figure Legend Snippet: Determination of AvBD8 protein expression in immune cell lines by performing immunocytochemical analysis. Cultured chicken CU91 T-, DT40 B-, HD11 macrophage-, and OU2 fibroblast-cell lines were incubated with the rabbit anti-AvBD8 primary antibody, followed by incubation with the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody, and were counterstained with 4′,6-diamidino-2-phenylindole. Control cells were incubated with secondary antibody only. AvBD8, avian beta-defensin 8; IgG, immunoglobulin G. Scale bar: 20 μm.

    Techniques Used: Expressing, Cell Culture, Incubation

    Determination of AvBD8 protein expression in immune tissues of male WL chickens by performing immunohistochemical analysis. Immunohistochemical analysis was performed to assess AvBD8 protein expression in the thymus, spleen, liver, small intestine, and ceca of male WL chickens aged 25 weeks. Frozen sections were incubated with the rabbit anti-AvBD8 primary antibody, followed by incubation with the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody, and were counterstained with 4′,6-diamidino-2-phenylindole. Control sections were incubated with secondary antibody only. Boxed region in the middle column is enlarged in the right column. AvBD8, avian beta-defensin 8; WL, White Leghorn; IgG, immunoglobulin G. Scale bar: 200 μm (left and middle columns) and 50 μm (right column). Arrowheads indicate strong AvBD8 signal in the intestinal mucosal layer.
    Figure Legend Snippet: Determination of AvBD8 protein expression in immune tissues of male WL chickens by performing immunohistochemical analysis. Immunohistochemical analysis was performed to assess AvBD8 protein expression in the thymus, spleen, liver, small intestine, and ceca of male WL chickens aged 25 weeks. Frozen sections were incubated with the rabbit anti-AvBD8 primary antibody, followed by incubation with the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody, and were counterstained with 4′,6-diamidino-2-phenylindole. Control sections were incubated with secondary antibody only. Boxed region in the middle column is enlarged in the right column. AvBD8, avian beta-defensin 8; WL, White Leghorn; IgG, immunoglobulin G. Scale bar: 200 μm (left and middle columns) and 50 μm (right column). Arrowheads indicate strong AvBD8 signal in the intestinal mucosal layer.

    Techniques Used: Expressing, Immunohistochemistry, Incubation

    53) Product Images from "Triple blockade of EGFR, MEK and PD-L1 has antitumor activity in colorectal cancer models with constitutive activation of MAPK signaling and PD-L1 overexpression"

    Article Title: Triple blockade of EGFR, MEK and PD-L1 has antitumor activity in colorectal cancer models with constitutive activation of MAPK signaling and PD-L1 overexpression

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-019-1497-0

    Expression of PD-L1 and EGFR by immunofluorescence analysis. a. Immunofluorescence analysis of the localization of EGFR and PD-L1 in HCT116 and HCT116-MR cells after exposure to different time of TGFα (5 min, 20 min and 1 h). Cells were stained with anti-EGFR antibody followed by secondary antibody labeled with Alexa Fluor 488 and anti-PD-L1 antibody followed by secondary antibody Alexa Fluor 532. DAPI was included to stain the nucleus. b. Cytofluorimetric analysis of HCT116 MR cells. Cells are stimulated with TGFα for 5 min, 20 min and 1 h alone or in combination with MEKi (BAY86–9766 0,5 μM) and then we investigated the presence of EGFR and PD-L1 on the surface of cells. c. An upregulation of PD-L1 is observed in the nucleus of HCT116 MR cells. Green color from Alexa Fluor 488 for PD-L1 and blue color from DAPI for visualization of nuclei. d. HCT116 and HCT116-MR were lysed to obtain cytosolic and nuclear fragments that were analyzed by Western blot analysis. Tubulin and Histone H3 antibodies were used as loading and purity control of the cytosolic and nuclear fractions, respectively
    Figure Legend Snippet: Expression of PD-L1 and EGFR by immunofluorescence analysis. a. Immunofluorescence analysis of the localization of EGFR and PD-L1 in HCT116 and HCT116-MR cells after exposure to different time of TGFα (5 min, 20 min and 1 h). Cells were stained with anti-EGFR antibody followed by secondary antibody labeled with Alexa Fluor 488 and anti-PD-L1 antibody followed by secondary antibody Alexa Fluor 532. DAPI was included to stain the nucleus. b. Cytofluorimetric analysis of HCT116 MR cells. Cells are stimulated with TGFα for 5 min, 20 min and 1 h alone or in combination with MEKi (BAY86–9766 0,5 μM) and then we investigated the presence of EGFR and PD-L1 on the surface of cells. c. An upregulation of PD-L1 is observed in the nucleus of HCT116 MR cells. Green color from Alexa Fluor 488 for PD-L1 and blue color from DAPI for visualization of nuclei. d. HCT116 and HCT116-MR were lysed to obtain cytosolic and nuclear fragments that were analyzed by Western blot analysis. Tubulin and Histone H3 antibodies were used as loading and purity control of the cytosolic and nuclear fractions, respectively

    Techniques Used: Expressing, Immunofluorescence, Staining, Labeling, Western Blot

    54) Product Images from "Neoglycoconjugate of Tetrasaccharide Representing One Repeating Unit of the Streptococcus pneumoniae Type 14 Capsular Polysaccharide Induces the Production of Opsonizing IgG1 Antibodies and Possesses the Highest Protective Activity As Compared to Hexa- and Octasaccharide Conjugates"

    Article Title: Neoglycoconjugate of Tetrasaccharide Representing One Repeating Unit of the Streptococcus pneumoniae Type 14 Capsular Polysaccharide Induces the Production of Opsonizing IgG1 Antibodies and Possesses the Highest Protective Activity As Compared to Hexa- and Octasaccharide Conjugates

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.00659

    Inhibition of IgG1 antibodies (Abs) in mice immune sera with OS ligands and CP. Enzyme-linked immunosorbent assay inhibition assays were performed using streptavidin-coated plates with tetra-biotin, hexa-biotin, and octa-biotin adsorbed on their surfaces. (A–C) Inhibition of IgG1 Abs in the pooled sera of BALB/c mice ( n = 6) that were immunized twice intraperitoneally with glycoconjugates at 10 µg/dose. Sera were obtained 14 days after the second immunization. Serum samples for all glycoconjugates were diluted 1:4,000. The tetra-, hexa-, and octasaccharide ligands, synCP, and bacCP were used as inhibitors and applied in amounts ranging from 0 to 10 µg/well. The horizontal line indicates the IC 50 at the point of intersection of the inhibition curves. (A) Tetra-bovine serum albumin (BSA) conjugate antiserum was tested against tetra-biotin capture material. (B) Hexa-BSA conjugate antiserum was tested against hexa-biotin capture material (C) . Octa-BSA conjugate antiserum was tested against octa-biotin capture material. (D) Inhibition of IgG1 Abs was measured in the pooled sera of BALB/c mice immunized twice intraperitoneally over 2 weeks with the conjugated pneumococcal vaccine, Prevenar-13, at 1.1 µg of Streptococcus pneumoniae type 14 CP per single dose. The dilutions of sera tested against the tetra-biotin and octa-biotin coating antigens were 1:500; hexa-biotin was diluted 1:300. (E) Inhibition of IgG Abs was measured in serum harvested from rabbits that were immunized multiple times with inactivated S. pneumonia type 14 bacteria. The dilution of rabbit sera tested against tetra-biotin coating antigens was 1:300, against hexa-biotin and octa-biotin coating antigens was 1:3,000; n = 3 per data point.
    Figure Legend Snippet: Inhibition of IgG1 antibodies (Abs) in mice immune sera with OS ligands and CP. Enzyme-linked immunosorbent assay inhibition assays were performed using streptavidin-coated plates with tetra-biotin, hexa-biotin, and octa-biotin adsorbed on their surfaces. (A–C) Inhibition of IgG1 Abs in the pooled sera of BALB/c mice ( n = 6) that were immunized twice intraperitoneally with glycoconjugates at 10 µg/dose. Sera were obtained 14 days after the second immunization. Serum samples for all glycoconjugates were diluted 1:4,000. The tetra-, hexa-, and octasaccharide ligands, synCP, and bacCP were used as inhibitors and applied in amounts ranging from 0 to 10 µg/well. The horizontal line indicates the IC 50 at the point of intersection of the inhibition curves. (A) Tetra-bovine serum albumin (BSA) conjugate antiserum was tested against tetra-biotin capture material. (B) Hexa-BSA conjugate antiserum was tested against hexa-biotin capture material (C) . Octa-BSA conjugate antiserum was tested against octa-biotin capture material. (D) Inhibition of IgG1 Abs was measured in the pooled sera of BALB/c mice immunized twice intraperitoneally over 2 weeks with the conjugated pneumococcal vaccine, Prevenar-13, at 1.1 µg of Streptococcus pneumoniae type 14 CP per single dose. The dilutions of sera tested against the tetra-biotin and octa-biotin coating antigens were 1:500; hexa-biotin was diluted 1:300. (E) Inhibition of IgG Abs was measured in serum harvested from rabbits that were immunized multiple times with inactivated S. pneumonia type 14 bacteria. The dilution of rabbit sera tested against tetra-biotin coating antigens was 1:300, against hexa-biotin and octa-biotin coating antigens was 1:3,000; n = 3 per data point.

    Techniques Used: Inhibition, Mouse Assay, Enzyme-linked Immunosorbent Assay

    Anti-CP IgG1 antibody (Ab) titers in mice immunized with the glycoconjugates. BALB/c mice were immunized intraperitoneally with tetra-bovine serum albumin (BSA) (A) , hexa-BSA (B) , and octa-BSA (C) conjugates adsorbed on aluminum hydroxide twice over 14 days at 1.25–10 µg/dose (the hexa-BSA conjugate was injected at 2.5–10 µg/dose). Anti-CP IgG1 Ab titers in murine blood sera were determined by enzyme-linked immunosorbent assay 2 weeks after the second immunization. Streptococcus pneumoniae type 14 bacterial CP was used as the coating antigens. The data from two experiments were summarized. For each glycoconjugate, blood was taken from 6 to 12 mice. The data represent individual anti-CP IgG1 Ab titers, bars indicates median ± SD. Mann–Whitney Rank Sum tests were used to evaluate significance. Differences in the anti-CP IgG1 Ab titers between tetra-BSA and octa-BSA conjugates at the immunizing dose of 10 µg/mouse, * P
    Figure Legend Snippet: Anti-CP IgG1 antibody (Ab) titers in mice immunized with the glycoconjugates. BALB/c mice were immunized intraperitoneally with tetra-bovine serum albumin (BSA) (A) , hexa-BSA (B) , and octa-BSA (C) conjugates adsorbed on aluminum hydroxide twice over 14 days at 1.25–10 µg/dose (the hexa-BSA conjugate was injected at 2.5–10 µg/dose). Anti-CP IgG1 Ab titers in murine blood sera were determined by enzyme-linked immunosorbent assay 2 weeks after the second immunization. Streptococcus pneumoniae type 14 bacterial CP was used as the coating antigens. The data from two experiments were summarized. For each glycoconjugate, blood was taken from 6 to 12 mice. The data represent individual anti-CP IgG1 Ab titers, bars indicates median ± SD. Mann–Whitney Rank Sum tests were used to evaluate significance. Differences in the anti-CP IgG1 Ab titers between tetra-BSA and octa-BSA conjugates at the immunizing dose of 10 µg/mouse, * P

    Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    IgG1 antibody (Ab) titer in mice immunized with Streptococcus pneumoniae type 14 CP. BALB/c mice received two intraperitoneal immunizations with the conjugated pneumococcal vaccine Prevenar-13 with aluminum phosphate as adjuvant at 1.1 µg (content of S. pneumoniae type 14 CP) per dose. The tetra-bovine serum albumin (BSA), hexa-BSA, and octa-BSA conjugates, and synthetic (synCP) and bacterial CP (bacCP) were capture antigens, coating the enzyme-linked immunosorbent assay plates. The data are represented by individual titers of IgG1 Abs, bars indicate median ± SD. Mann–Whitney Rank Sum tests were used to determine significance. For differences in the level of Abs detected against tetra-BSA and octa-BSA conjugates, * P
    Figure Legend Snippet: IgG1 antibody (Ab) titer in mice immunized with Streptococcus pneumoniae type 14 CP. BALB/c mice received two intraperitoneal immunizations with the conjugated pneumococcal vaccine Prevenar-13 with aluminum phosphate as adjuvant at 1.1 µg (content of S. pneumoniae type 14 CP) per dose. The tetra-bovine serum albumin (BSA), hexa-BSA, and octa-BSA conjugates, and synthetic (synCP) and bacterial CP (bacCP) were capture antigens, coating the enzyme-linked immunosorbent assay plates. The data are represented by individual titers of IgG1 Abs, bars indicate median ± SD. Mann–Whitney Rank Sum tests were used to determine significance. For differences in the level of Abs detected against tetra-BSA and octa-BSA conjugates, * P

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    Anti-OS IgG1 antibody (Ab) titer in mice immunized with the OS-conjugates, CP, or bacteria. The biotinylated oligosaccharides tetra-biotin, hexa-biotin, and octa-biotin were applied as coating antigens. (A) Fourteen days after the second immunization, IgG1 Ab titers were determined in the pooled sera of BALB/c mice ( n = 6) intraperitoneally vaccinated with a single dose (10 µg) of each conjugate adsorbed on aluminum hydroxide. (B) Fourteen days after the second immunization, the IgG1 Ab titers were measured in the pooled sera of two groups of BALB/c mice ( n = 6) who were intraperitoneally vaccinated with single doses of either 1.1 or 2.2 µg (CP content) of Streptococcus pneumoniae type 14 CP-CPM 197 adsorbed on aluminum phosphate. (C) The level of IgG Abs in rabbits ( n = 2) immunized with inactive S. pneumoniae type 14 bacterial cells. Ab titers were transformed to log 10 . n = 6 assays per antiserum. The data are displayed as a mean value ± SD. Mann–Whitney Rank Sum tests were used to determine significance, * P
    Figure Legend Snippet: Anti-OS IgG1 antibody (Ab) titer in mice immunized with the OS-conjugates, CP, or bacteria. The biotinylated oligosaccharides tetra-biotin, hexa-biotin, and octa-biotin were applied as coating antigens. (A) Fourteen days after the second immunization, IgG1 Ab titers were determined in the pooled sera of BALB/c mice ( n = 6) intraperitoneally vaccinated with a single dose (10 µg) of each conjugate adsorbed on aluminum hydroxide. (B) Fourteen days after the second immunization, the IgG1 Ab titers were measured in the pooled sera of two groups of BALB/c mice ( n = 6) who were intraperitoneally vaccinated with single doses of either 1.1 or 2.2 µg (CP content) of Streptococcus pneumoniae type 14 CP-CPM 197 adsorbed on aluminum phosphate. (C) The level of IgG Abs in rabbits ( n = 2) immunized with inactive S. pneumoniae type 14 bacterial cells. Ab titers were transformed to log 10 . n = 6 assays per antiserum. The data are displayed as a mean value ± SD. Mann–Whitney Rank Sum tests were used to determine significance, * P

    Techniques Used: Mouse Assay, Transformation Assay, MANN-WHITNEY

    Inhibition of IgG antibodies (Abs) recognizing SynCP as the coating antigen with OS ligands and CP. Pooled immune sera were obtained after double intraperitoneal immunization of BALB/c mice ( n = 6 for each glycoconjugate) with the OS-bovine serum albumin (BSA) conjugates adsorbed on aluminum hydroxide (10 µg of carbohydrate/single dose). The tetra-, hexa-, and octasaccharide ligands, as well as synCP and bacCP, were used as inhibitory materials at concentrations of 0–10 µg/well. The horizontal line indicates the IC 50 level at the point of intersection of the inhibition curves. (A) Inhibition of IgG Abs was measured in tetra-BSA conjugate sera at a dilution of 1:400. (B) Inhibition of IgG Abs was measured in hexa-BSA conjugate sera at a dilution of 1:400. (C) Inhibition of IgG Abs was measured in octa-BSA conjugate sera at a dilution of 1:1,600. (D) Inhibition of IgG Abs was measured in Streptococcus pneumoniae type 14 CP-CRM 197 at a dilution of 1:200. Immune sera were obtained after immunization of mice ( n = 6) with Prevenar-13 adsorbed on aluminum phosphate at 2.2 µg (content of S. pneumoniae type 14 CP) per single dose.
    Figure Legend Snippet: Inhibition of IgG antibodies (Abs) recognizing SynCP as the coating antigen with OS ligands and CP. Pooled immune sera were obtained after double intraperitoneal immunization of BALB/c mice ( n = 6 for each glycoconjugate) with the OS-bovine serum albumin (BSA) conjugates adsorbed on aluminum hydroxide (10 µg of carbohydrate/single dose). The tetra-, hexa-, and octasaccharide ligands, as well as synCP and bacCP, were used as inhibitory materials at concentrations of 0–10 µg/well. The horizontal line indicates the IC 50 level at the point of intersection of the inhibition curves. (A) Inhibition of IgG Abs was measured in tetra-BSA conjugate sera at a dilution of 1:400. (B) Inhibition of IgG Abs was measured in hexa-BSA conjugate sera at a dilution of 1:400. (C) Inhibition of IgG Abs was measured in octa-BSA conjugate sera at a dilution of 1:1,600. (D) Inhibition of IgG Abs was measured in Streptococcus pneumoniae type 14 CP-CRM 197 at a dilution of 1:200. Immune sera were obtained after immunization of mice ( n = 6) with Prevenar-13 adsorbed on aluminum phosphate at 2.2 µg (content of S. pneumoniae type 14 CP) per single dose.

    Techniques Used: Inhibition, Mouse Assay

    55) Product Images from "Transcriptional repression of human epidermal growth factor receptor 2 by ClC‐3 Cl−/H+ transporter inhibition in human breast cancer cells. Transcriptional repression of human epidermal growth factor receptor 2 by ClC‐3 Cl−/H+ transporter inhibition in human breast cancer cells"

    Article Title: Transcriptional repression of human epidermal growth factor receptor 2 by ClC‐3 Cl−/H+ transporter inhibition in human breast cancer cells. Transcriptional repression of human epidermal growth factor receptor 2 by ClC‐3 Cl−/H+ transporter inhibition in human breast cancer cells

    Journal: Cancer Science

    doi: 10.1111/cas.13715

    Cellular distribution of ClC‐3 in MDA‐MB‐453 cells. (A‐C) Immunocytochemical staining of MDA‐MB‐453 cells for ClC‐3. A, Confocal images of a cell labeled with anti‐ClC‐3 and Alexa Fluor 488 anti‐rabbit IgG antibodies. B, a fluorescent image merged with a transparent image. C, the profile of fluorescence intensity on the white dotted line in (B) is shown in “green”. The plasma membrane is shown in ‘red’. D‐H, ClC‐3 fluorescent images in control siRNA (si‐cont) (D)‐, ClC‐3 siRNA (si‐ClC‐3) (E)‐, ClC‐4 siRNA (si‐ClC‐4) (F)‐, and ClC‐5 siRNA (si‐ClC‐5)‐transfected MDA‐MB‐453 cells for 72 h. Mean fluorescent intensity in the si‐cont group was expressed as 1.0 (n = 24 for each); summarized data are shown in (H). Results are expressed as means ± SEM. ** P
    Figure Legend Snippet: Cellular distribution of ClC‐3 in MDA‐MB‐453 cells. (A‐C) Immunocytochemical staining of MDA‐MB‐453 cells for ClC‐3. A, Confocal images of a cell labeled with anti‐ClC‐3 and Alexa Fluor 488 anti‐rabbit IgG antibodies. B, a fluorescent image merged with a transparent image. C, the profile of fluorescence intensity on the white dotted line in (B) is shown in “green”. The plasma membrane is shown in ‘red’. D‐H, ClC‐3 fluorescent images in control siRNA (si‐cont) (D)‐, ClC‐3 siRNA (si‐ClC‐3) (E)‐, ClC‐4 siRNA (si‐ClC‐4) (F)‐, and ClC‐5 siRNA (si‐ClC‐5)‐transfected MDA‐MB‐453 cells for 72 h. Mean fluorescent intensity in the si‐cont group was expressed as 1.0 (n = 24 for each); summarized data are shown in (H). Results are expressed as means ± SEM. ** P

    Techniques Used: Multiple Displacement Amplification, Staining, Labeling, Fluorescence, Transfection

    56) Product Images from "A Microfluidic Immunostaining System Enables Quality Assured and Standardized Immunohistochemical Biomarker Analysis"

    Article Title: A Microfluidic Immunostaining System Enables Quality Assured and Standardized Immunohistochemical Biomarker Analysis

    Journal: Scientific Reports

    doi: 10.1038/srep45968

    Comparison of the staining intensities between a batch process and a microfluidic process using HER2 Abs and QD605-IgG with SKBR3 cell sections. ( A , B ) Staining results using the microfluidic device at 5, 15, 30, and 60 min. The data are mean ± s.d.; n = 3–4. ( C ) HER2 Ab was labeled with QD605 using a microfluidic immunostaining system (10 min) and a batch staining process (2 h); the concentration ranged from 0.375 ng mL −1 to 24 ng mL −1 . ( D ) Quantitative analysis of the HER2 staining intensity between a batch process and microfluidic staining. The data are mean ± s.d.; n = 3–7. ( E ) Staining intensities of three HER2 Abs (Dako: 1.6 μg mL −1 , Abcam: 0.024 μg mL −1 , Thermo Fisher Scientific: 0.36 mg mL −1 [for 1.000×]) at several concentrations using a 2 h batch process and a 10 min microfluidic process. The data are mean ± s.d.; n = 3–8 for 2 h batch process and n = 3–4 for microfluidic process.
    Figure Legend Snippet: Comparison of the staining intensities between a batch process and a microfluidic process using HER2 Abs and QD605-IgG with SKBR3 cell sections. ( A , B ) Staining results using the microfluidic device at 5, 15, 30, and 60 min. The data are mean ± s.d.; n = 3–4. ( C ) HER2 Ab was labeled with QD605 using a microfluidic immunostaining system (10 min) and a batch staining process (2 h); the concentration ranged from 0.375 ng mL −1 to 24 ng mL −1 . ( D ) Quantitative analysis of the HER2 staining intensity between a batch process and microfluidic staining. The data are mean ± s.d.; n = 3–7. ( E ) Staining intensities of three HER2 Abs (Dako: 1.6 μg mL −1 , Abcam: 0.024 μg mL −1 , Thermo Fisher Scientific: 0.36 mg mL −1 [for 1.000×]) at several concentrations using a 2 h batch process and a 10 min microfluidic process. The data are mean ± s.d.; n = 3–8 for 2 h batch process and n = 3–4 for microfluidic process.

    Techniques Used: Staining, Labeling, Immunostaining, Concentration Assay

    Fluorescence images of QD605s in SKBR3 cell sections labeled with a form of quantum dot-conjugated IgG (QD-IgG). ( A ) Fluorescence images of QD605-conjugated goat anti-rabbit IgG incubated in a SKBR3 cell section using a microfluidic immunostaining system. ( B–D ) Comparison of fluorescence images stained using a microfluidic system (left) and a batch process (right). The incubation results from concentrations of 0.5×, 0.250×, and 0.125×. Red color shows the QD605-labeled cells and blue color comes from the autofluorescence of cell sections caused by the excitation of ultraviolet.
    Figure Legend Snippet: Fluorescence images of QD605s in SKBR3 cell sections labeled with a form of quantum dot-conjugated IgG (QD-IgG). ( A ) Fluorescence images of QD605-conjugated goat anti-rabbit IgG incubated in a SKBR3 cell section using a microfluidic immunostaining system. ( B–D ) Comparison of fluorescence images stained using a microfluidic system (left) and a batch process (right). The incubation results from concentrations of 0.5×, 0.250×, and 0.125×. Red color shows the QD605-labeled cells and blue color comes from the autofluorescence of cell sections caused by the excitation of ultraviolet.

    Techniques Used: Fluorescence, Labeling, Incubation, Immunostaining, Staining

    57) Product Images from "Transcriptional repression of human epidermal growth factor receptor 2 by ClC‐3 Cl−/H+ transporter inhibition in human breast cancer cells. Transcriptional repression of human epidermal growth factor receptor 2 by ClC‐3 Cl−/H+ transporter inhibition in human breast cancer cells"

    Article Title: Transcriptional repression of human epidermal growth factor receptor 2 by ClC‐3 Cl−/H+ transporter inhibition in human breast cancer cells. Transcriptional repression of human epidermal growth factor receptor 2 by ClC‐3 Cl−/H+ transporter inhibition in human breast cancer cells

    Journal: Cancer Science

    doi: 10.1111/cas.13715

    Cellular distribution of ClC‐3 in MDA‐MB‐453 cells. (A‐C) Immunocytochemical staining of MDA‐MB‐453 cells for ClC‐3. A, Confocal images of a cell labeled with anti‐ClC‐3 and Alexa Fluor 488 anti‐rabbit IgG antibodies. B, a fluorescent image merged with a transparent image. C, the profile of fluorescence intensity on the white dotted line in (B) is shown in “green”. The plasma membrane is shown in ‘red’. D‐H, ClC‐3 fluorescent images in control siRNA (si‐cont) (D)‐, ClC‐3 siRNA (si‐ClC‐3) (E)‐, ClC‐4 siRNA (si‐ClC‐4) (F)‐, and ClC‐5 siRNA (si‐ClC‐5)‐transfected MDA‐MB‐453 cells for 72 h. Mean fluorescent intensity in the si‐cont group was expressed as 1.0 (n = 24 for each); summarized data are shown in (H). Results are expressed as means ± SEM. ** P
    Figure Legend Snippet: Cellular distribution of ClC‐3 in MDA‐MB‐453 cells. (A‐C) Immunocytochemical staining of MDA‐MB‐453 cells for ClC‐3. A, Confocal images of a cell labeled with anti‐ClC‐3 and Alexa Fluor 488 anti‐rabbit IgG antibodies. B, a fluorescent image merged with a transparent image. C, the profile of fluorescence intensity on the white dotted line in (B) is shown in “green”. The plasma membrane is shown in ‘red’. D‐H, ClC‐3 fluorescent images in control siRNA (si‐cont) (D)‐, ClC‐3 siRNA (si‐ClC‐3) (E)‐, ClC‐4 siRNA (si‐ClC‐4) (F)‐, and ClC‐5 siRNA (si‐ClC‐5)‐transfected MDA‐MB‐453 cells for 72 h. Mean fluorescent intensity in the si‐cont group was expressed as 1.0 (n = 24 for each); summarized data are shown in (H). Results are expressed as means ± SEM. ** P

    Techniques Used: Multiple Displacement Amplification, Staining, Labeling, Fluorescence, Transfection

    58) Product Images from "Development and initial evaluation of a lateral flow dipstick test for antigen detection of Entamoeba histolytica in stool sample"

    Article Title: Development and initial evaluation of a lateral flow dipstick test for antigen detection of Entamoeba histolytica in stool sample

    Journal: Pathogens and Global Health

    doi: 10.1080/20477724.2017.1300421

    Western blot of E. histolytica rPPDK and native PPDK using anti-rPPDK IgG and anti-EhESA IgG polyclonal antibodies.
    Figure Legend Snippet: Western blot of E. histolytica rPPDK and native PPDK using anti-rPPDK IgG and anti-EhESA IgG polyclonal antibodies.

    Techniques Used: Western Blot

    59) Product Images from "Epigenetic priming restores the HLA class-I antigen processing machinery expression in Merkel cell carcinoma"

    Article Title: Epigenetic priming restores the HLA class-I antigen processing machinery expression in Merkel cell carcinoma

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-02608-0

    Reduced expression of antigen processing machinery genes is mediated by histone hypoacetylation and is increased by pharmacologic histone deactylase inhibition. For all experiments the indicated MCC cell lines were analyzed without treatment (grey) and after treatment with mithramycin A (MA, turquoise), vorinostat (V, light blue), or the combination thereof (V + MA, dark blue). ( A ) Global H3K9 acetylation of untreated, V, MA or V + MA treated MCC cell lines was determined by immunoblot with an AcH3K9 antibody; β-tubulin served as loading control. ( B ) Chromatin immunoprecipitation (ChIP) assay was performed with untreated or V + MA treated WaGa cells followed by a qRT-PCR using TAP1 , TAP2 , LMP2 or LMP7 promoter specific primers. C T values of AcH3K9 antibody or rabbit IgG isotype precipitated DNA were normalized to histone H3 antibody precipitated DNA as described in material and methods. Experiments were performed in triplicates twice and results are expressed as mean + SEM. ( C ) mRNA expression of HLA-A , B2M , TAP1 , TAP2 , LMP2 and LMP7 was determined by RT-qPCR in triplicates; C T values were normalized to RPLP0 and calibrated to the ΔC T value of untreated MKL-2 cells; relative mRNA expression is depicted + SEM for MKL-2, BroLi, MKL-1 and WaGa. ( D ) HLA class-I intracellular localization was determined via immunofluorescent staining using an HLA-A detecting antibody (clone EP1395Y) with a dylight 488 labeled secondary antibody (green). Wheat germ agglutinin (Alexa fluor 647, red) was used to stain cellular membranes and DAPI (blue) served as nuclear stain. Single cell images in the upper right corner of the overview image were acquired using confocal microscopy. ( E , F ) HLA class-I cell surface expression was determined by flow cytometry using a HLA-ABC specific antibody as exemplified for WaGa ( E ); the results for all cell lines analyzed are depicted as the geometric mean fluorescence intensity (gMFI) of HLA class-I (HLA-ABC) staining, + SEM in three independent experiments ( F ). Statistical analysis was performed using the Friedman test as indicated. ( G ) Viral protein expression was determined by immunoblot of whole cell lysates using an antibody specific for LT (clone CM2B4) depicting the different truncated LTs characteristically for MCC cells. β-tubulin served as loading control.
    Figure Legend Snippet: Reduced expression of antigen processing machinery genes is mediated by histone hypoacetylation and is increased by pharmacologic histone deactylase inhibition. For all experiments the indicated MCC cell lines were analyzed without treatment (grey) and after treatment with mithramycin A (MA, turquoise), vorinostat (V, light blue), or the combination thereof (V + MA, dark blue). ( A ) Global H3K9 acetylation of untreated, V, MA or V + MA treated MCC cell lines was determined by immunoblot with an AcH3K9 antibody; β-tubulin served as loading control. ( B ) Chromatin immunoprecipitation (ChIP) assay was performed with untreated or V + MA treated WaGa cells followed by a qRT-PCR using TAP1 , TAP2 , LMP2 or LMP7 promoter specific primers. C T values of AcH3K9 antibody or rabbit IgG isotype precipitated DNA were normalized to histone H3 antibody precipitated DNA as described in material and methods. Experiments were performed in triplicates twice and results are expressed as mean + SEM. ( C ) mRNA expression of HLA-A , B2M , TAP1 , TAP2 , LMP2 and LMP7 was determined by RT-qPCR in triplicates; C T values were normalized to RPLP0 and calibrated to the ΔC T value of untreated MKL-2 cells; relative mRNA expression is depicted + SEM for MKL-2, BroLi, MKL-1 and WaGa. ( D ) HLA class-I intracellular localization was determined via immunofluorescent staining using an HLA-A detecting antibody (clone EP1395Y) with a dylight 488 labeled secondary antibody (green). Wheat germ agglutinin (Alexa fluor 647, red) was used to stain cellular membranes and DAPI (blue) served as nuclear stain. Single cell images in the upper right corner of the overview image were acquired using confocal microscopy. ( E , F ) HLA class-I cell surface expression was determined by flow cytometry using a HLA-ABC specific antibody as exemplified for WaGa ( E ); the results for all cell lines analyzed are depicted as the geometric mean fluorescence intensity (gMFI) of HLA class-I (HLA-ABC) staining, + SEM in three independent experiments ( F ). Statistical analysis was performed using the Friedman test as indicated. ( G ) Viral protein expression was determined by immunoblot of whole cell lysates using an antibody specific for LT (clone CM2B4) depicting the different truncated LTs characteristically for MCC cells. β-tubulin served as loading control.

    Techniques Used: Expressing, Inhibition, Chromatin Immunoprecipitation, Quantitative RT-PCR, Staining, Labeling, Confocal Microscopy, Flow Cytometry, Cytometry, Fluorescence

    60) Product Images from "Distribution and localization of fibroblast growth factor-8 in rat brain and nerve cells during neural stem/progenitor cell differentiation ☆"

    Article Title: Distribution and localization of fibroblast growth factor-8 in rat brain and nerve cells during neural stem/progenitor cell differentiation ☆

    Journal: Neural Regeneration Research

    doi: 10.3969/j.issn.1673-5374.2012.19.002

    Immunofluorescence analyses of fibroblast growth factor-8 (FGF-8) and fibroblast growth factor receptor-3 (FGFR-3) localization in cells during neural stem/progenitor cells (NSCs/NPCs) differentiation (confocal microscope). (A–D) FGF-8 + (green, FITC); (E-H) FGFR-3 + (green, FITC). Green: FITC, FGF-8 + or FGFR-3 + ; red: TRITC, nestin + ; blue: DAPI, nuclei; multicolor: merged. (C, D, G, H) Triangle arrows indicate positive staining in the cytoplasm in permeable treatment groups. Thin arrows indicate negative staining. (B, F) Large arrows indicate positive staining on the plasma membranes of differentiated cells in NSCs/NPCs in non-permeable treatment groups. Scale bars: (A, E) 50 μm; (B, C, F, G) 100 μm; (D, H) 20 μm. n = 4. Non-PTG: non-permeable treatment group; PTG: permeable treatment group. FITC: Fluorescein isothiocyanate; TRITC: tetraethylrhodamine isothiocyanate.
    Figure Legend Snippet: Immunofluorescence analyses of fibroblast growth factor-8 (FGF-8) and fibroblast growth factor receptor-3 (FGFR-3) localization in cells during neural stem/progenitor cells (NSCs/NPCs) differentiation (confocal microscope). (A–D) FGF-8 + (green, FITC); (E-H) FGFR-3 + (green, FITC). Green: FITC, FGF-8 + or FGFR-3 + ; red: TRITC, nestin + ; blue: DAPI, nuclei; multicolor: merged. (C, D, G, H) Triangle arrows indicate positive staining in the cytoplasm in permeable treatment groups. Thin arrows indicate negative staining. (B, F) Large arrows indicate positive staining on the plasma membranes of differentiated cells in NSCs/NPCs in non-permeable treatment groups. Scale bars: (A, E) 50 μm; (B, C, F, G) 100 μm; (D, H) 20 μm. n = 4. Non-PTG: non-permeable treatment group; PTG: permeable treatment group. FITC: Fluorescein isothiocyanate; TRITC: tetraethylrhodamine isothiocyanate.

    Techniques Used: Immunofluorescence, Microscopy, Staining, Negative Staining

    61) Product Images from "The sickle cell trait affects contact dynamics and endothelial cell activation in Plasmodium falciparum-infected erythrocytes"

    Article Title: The sickle cell trait affects contact dynamics and endothelial cell activation in Plasmodium falciparum-infected erythrocytes

    Journal: Communications Biology

    doi: 10.1038/s42003-018-0223-3

    Reduced endothelial cell activation by infected HbAS erythrocytes. a Representative confocal microscopy images showing the subcellular localization of p65-NFĸB subunit in human dermal microvascular endothelial cells (HDMEC) upon TNF-α treatment (100 units ml −1 for 2 h) or upon co-culture with parasitized HbAA or HbAS erythrocytes (1 × 10 7 infected red blood cells at the trophozoite stage for 2 h) under static conditions. Medium served as a negative control. The p65-NFĸB subunit was localized using a specific polyclonal rabbit antiserum (dilution 1:200) and an Alexa-488 conjugated goat anti-rabbit IgG antiserum (1:400) as secondary antibody. The nuclei were stained using Hoechst 33342. Scale bar, 20 µm. b Percentage of HDMECs positive for nuclear p65-NFĸB staining (nuclear labeling index, NLI) as a function of the parasite load under static conditions. Infected HbAA and HbAS erythrocytes (iHbAA and iHbAS) were analyzed. TNF-α served as a positive control and uninfected HbAA and HbAS red blood cells (HbAA and HbAS) were used as negative controls. As an additional control, the effect of an isogenic knobless FCR3 line (iHbAA K−) was investigated. The mean ± SEM of at least three independent biological replicates is shown, with at least 50 endothelial cells being analyzed per condition and per biological replicate. Note the data obtained for parasitized HbAA and HbAS as well as for the HbAA erythrocytes infected with the knobless parasite line are significantly different, as determined using F -tests ( F = 28; DF = 4; p
    Figure Legend Snippet: Reduced endothelial cell activation by infected HbAS erythrocytes. a Representative confocal microscopy images showing the subcellular localization of p65-NFĸB subunit in human dermal microvascular endothelial cells (HDMEC) upon TNF-α treatment (100 units ml −1 for 2 h) or upon co-culture with parasitized HbAA or HbAS erythrocytes (1 × 10 7 infected red blood cells at the trophozoite stage for 2 h) under static conditions. Medium served as a negative control. The p65-NFĸB subunit was localized using a specific polyclonal rabbit antiserum (dilution 1:200) and an Alexa-488 conjugated goat anti-rabbit IgG antiserum (1:400) as secondary antibody. The nuclei were stained using Hoechst 33342. Scale bar, 20 µm. b Percentage of HDMECs positive for nuclear p65-NFĸB staining (nuclear labeling index, NLI) as a function of the parasite load under static conditions. Infected HbAA and HbAS erythrocytes (iHbAA and iHbAS) were analyzed. TNF-α served as a positive control and uninfected HbAA and HbAS red blood cells (HbAA and HbAS) were used as negative controls. As an additional control, the effect of an isogenic knobless FCR3 line (iHbAA K−) was investigated. The mean ± SEM of at least three independent biological replicates is shown, with at least 50 endothelial cells being analyzed per condition and per biological replicate. Note the data obtained for parasitized HbAA and HbAS as well as for the HbAA erythrocytes infected with the knobless parasite line are significantly different, as determined using F -tests ( F = 28; DF = 4; p

    Techniques Used: Activation Assay, Infection, Confocal Microscopy, Co-Culture Assay, Negative Control, Staining, Labeling, Positive Control

    62) Product Images from "A Neuron-Optimized CRISPR/dCas9 Activation System for Robust and Specific Gene Regulation"

    Article Title: A Neuron-Optimized CRISPR/dCas9 Activation System for Robust and Specific Gene Regulation

    Journal: eNeuro

    doi: 10.1523/ENEURO.0495-18.2019

    CRISPRa-mediated induction of Fosb is neuron-selective in vivo . A , B , IHC performed for ( A ) NeuN or ( B ) GFAP alongside Fosb demonstrates neuronal selectivity of CRISPRa-mediated Fosb induction. Scale bar = 50 μm. C , Pixel density quantification and cross-correlation analysis reveals a signal overlap between Fosb and NeuN and depletion of signal between Fosb and GFAP ( n = 2 animals with eight regions of interest). All data are expressed as mean ± SEM.
    Figure Legend Snippet: CRISPRa-mediated induction of Fosb is neuron-selective in vivo . A , B , IHC performed for ( A ) NeuN or ( B ) GFAP alongside Fosb demonstrates neuronal selectivity of CRISPRa-mediated Fosb induction. Scale bar = 50 μm. C , Pixel density quantification and cross-correlation analysis reveals a signal overlap between Fosb and NeuN and depletion of signal between Fosb and GFAP ( n = 2 animals with eight regions of interest). All data are expressed as mean ± SEM.

    Techniques Used: In Vivo, Immunohistochemistry

    63) Product Images from "Selective Restoration of Pomc Expression in Glutamatergic POMC Neurons: Evidence for a Dynamic Hypothalamic Neurotransmitter Network"

    Article Title: Selective Restoration of Pomc Expression in Glutamatergic POMC Neurons: Evidence for a Dynamic Hypothalamic Neurotransmitter Network

    Journal: eNeuro

    doi: 10.1523/ENEURO.0400-18.2019

    IHC for POMC cell counts in control and restored mice, and from VGlut2-Cre; tdTomato animals. A , POMC-IR in a male control mouse detected with an Alexa Fluor 568 (red) secondary antibody (1:500). B , POMC-IR in a male restored mouse detected with an Alexa Fluor 568 (red) secondary antibody (1:500). C , POMC-IR in a female control mouse detected with biotinylated secondary antibody (1:500) and visualized with a diaminobenzidine (DAB) reaction (brown). D , POMC-IR in a female restored mouse detected with biotinylated secondary antibody (1:500) and visualized with a DAB reaction (brown). E , POMC-IR in a female Vglut2-Cre; tdTomato mouse detected with an Alexa Fluor 488 (green) secondary antibody (1:500; mirrored section from Fig. 1 G , H ). F , POMC neuron cell counts from sections (three per mouse). There was no difference between control (blue bars) or restored (green bars) mice, but only in the method of secondary labeling used. Male data for each group represented by filled blue circles and female data shown by filled pink circles; **** p
    Figure Legend Snippet: IHC for POMC cell counts in control and restored mice, and from VGlut2-Cre; tdTomato animals. A , POMC-IR in a male control mouse detected with an Alexa Fluor 568 (red) secondary antibody (1:500). B , POMC-IR in a male restored mouse detected with an Alexa Fluor 568 (red) secondary antibody (1:500). C , POMC-IR in a female control mouse detected with biotinylated secondary antibody (1:500) and visualized with a diaminobenzidine (DAB) reaction (brown). D , POMC-IR in a female restored mouse detected with biotinylated secondary antibody (1:500) and visualized with a DAB reaction (brown). E , POMC-IR in a female Vglut2-Cre; tdTomato mouse detected with an Alexa Fluor 488 (green) secondary antibody (1:500; mirrored section from Fig. 1 G , H ). F , POMC neuron cell counts from sections (three per mouse). There was no difference between control (blue bars) or restored (green bars) mice, but only in the method of secondary labeling used. Male data for each group represented by filled blue circles and female data shown by filled pink circles; **** p

    Techniques Used: Immunohistochemistry, Mouse Assay, Labeling

    Dual-label ISH for Pomc and Vglut2 or Gad67 , and relative Pomc expression in the medial basal hypothalamus of control and restored mice. A , ISH for Vglut2 (silver grains) and Pomc (red) in a female control mouse. Note that the green fluorescence of Alexa Fluor 488 used to detect Pomc was pseudocolored to red for these images. B , ISH for Gad67 (silver grains) and Pomc (red) in a female control mouse. C , ISH for Vglut2 (silver grains) and Pomc (red) in a female restored mouse. D , ISH for Gad67 (silver grains) and Pomc (red) in a female restored mouse. In panels A–D , blue arrows indicate overlap between Pomc and the silver grain ( Vglut2 or Gad67 ) signal. E , Degree of overlap between Pomc and Vglut2 (white bars) or Gad67 (grey bars) in control and restored mice, each animal’s Vlgut2/Pomc and Gad67/Pomc overlap percentage is connected by the solid black lines. F , Cell count of overlap between Pomc and Vglut2 (white bars) or Gad67 (grey bars) in control and restored mice, each animal’s Vlgut2/Pomc and Gad67/Pomc overlap count is connected by the solid black lines. G , Relative qRT-PCR of Pomc expression in the medial-basal hypothalamus of control (blue bar, left), FNΔ2 (red bar, middle), and restored (green bar, right) mice. Male data are represented by filled blue circles and female data by filled pink circles.
    Figure Legend Snippet: Dual-label ISH for Pomc and Vglut2 or Gad67 , and relative Pomc expression in the medial basal hypothalamus of control and restored mice. A , ISH for Vglut2 (silver grains) and Pomc (red) in a female control mouse. Note that the green fluorescence of Alexa Fluor 488 used to detect Pomc was pseudocolored to red for these images. B , ISH for Gad67 (silver grains) and Pomc (red) in a female control mouse. C , ISH for Vglut2 (silver grains) and Pomc (red) in a female restored mouse. D , ISH for Gad67 (silver grains) and Pomc (red) in a female restored mouse. In panels A–D , blue arrows indicate overlap between Pomc and the silver grain ( Vglut2 or Gad67 ) signal. E , Degree of overlap between Pomc and Vglut2 (white bars) or Gad67 (grey bars) in control and restored mice, each animal’s Vlgut2/Pomc and Gad67/Pomc overlap percentage is connected by the solid black lines. F , Cell count of overlap between Pomc and Vglut2 (white bars) or Gad67 (grey bars) in control and restored mice, each animal’s Vlgut2/Pomc and Gad67/Pomc overlap count is connected by the solid black lines. G , Relative qRT-PCR of Pomc expression in the medial-basal hypothalamus of control (blue bar, left), FNΔ2 (red bar, middle), and restored (green bar, right) mice. Male data are represented by filled blue circles and female data by filled pink circles.

    Techniques Used: In Situ Hybridization, Expressing, Mouse Assay, Fluorescence, Cell Counting, Quantitative RT-PCR

    Triple-label ISH for Pomc ( A , E , I ), Gad67 ( B , F , J ), Vglut2 (C,G,K), and overlaid signals ( D , H , L ) in WT mice throughout the rostral-caudal ARC axis. A–D , Low-magnification image of ISH signal for Pomc , Gad67 , Vglut2 , and the overlay of all signals from a male mouse. Note that the green fluorescence of Alexa Fluor 488 used to detect Pomc was pseudocolored to blue for these images. E–H , 40× images from the rostral ARC from a male mouse with Pomc neuron profiles outlined in yellow. I–L , 40× images from the caudal ARC from a female mouse with Pomc neuron profiles outlined in yellow. V indicates Vglut2 + Pomc neurons, G indicates Gad67 + Pomc neurons, and VG indicates Vglut2/Gad67 + Pomc neurons. M , The distribution of Pomc neurons along the rostral-caudal ARC axis. N , The overall percentages of Pomc -only (blue bar with filled inverted triangles), Gad67 + (red bar with filled squares), Vglut2 + (grey bar with filled circles), and Vglut2/Gad67 + (purple bar with filled triangles) Pomc neurons in the arcuate nucleus. Male data are represented by filled blue symbols and female data by filled pink symbols. O , The percentages of Pomc -only (blue line with filled inverted triangles), Gad67 + (red line with filled squares), Vglut2 + (grey line with filled circles), and Vglut2/Gad67 + (purple line with filled triangles) Pomc neurons at each coronal level 1 to 5 along the rostral-caudal ARC axis. P , Linear regression analysis of the relative percentage of each phenotypic category of Pomc neurons along the rostral-caudal ARC axis [ Pomc -only solid blue line, Gad67 + dotted red line, Gad67 + (levels 2–5) solid red line, Vglut2 + solid grey line, Vglut2/Gad67 + solid purple line].
    Figure Legend Snippet: Triple-label ISH for Pomc ( A , E , I ), Gad67 ( B , F , J ), Vglut2 (C,G,K), and overlaid signals ( D , H , L ) in WT mice throughout the rostral-caudal ARC axis. A–D , Low-magnification image of ISH signal for Pomc , Gad67 , Vglut2 , and the overlay of all signals from a male mouse. Note that the green fluorescence of Alexa Fluor 488 used to detect Pomc was pseudocolored to blue for these images. E–H , 40× images from the rostral ARC from a male mouse with Pomc neuron profiles outlined in yellow. I–L , 40× images from the caudal ARC from a female mouse with Pomc neuron profiles outlined in yellow. V indicates Vglut2 + Pomc neurons, G indicates Gad67 + Pomc neurons, and VG indicates Vglut2/Gad67 + Pomc neurons. M , The distribution of Pomc neurons along the rostral-caudal ARC axis. N , The overall percentages of Pomc -only (blue bar with filled inverted triangles), Gad67 + (red bar with filled squares), Vglut2 + (grey bar with filled circles), and Vglut2/Gad67 + (purple bar with filled triangles) Pomc neurons in the arcuate nucleus. Male data are represented by filled blue symbols and female data by filled pink symbols. O , The percentages of Pomc -only (blue line with filled inverted triangles), Gad67 + (red line with filled squares), Vglut2 + (grey line with filled circles), and Vglut2/Gad67 + (purple line with filled triangles) Pomc neurons at each coronal level 1 to 5 along the rostral-caudal ARC axis. P , Linear regression analysis of the relative percentage of each phenotypic category of Pomc neurons along the rostral-caudal ARC axis [ Pomc -only solid blue line, Gad67 + dotted red line, Gad67 + (levels 2–5) solid red line, Vglut2 + solid grey line, Vglut2/Gad67 + solid purple line].

    Techniques Used: In Situ Hybridization, Mouse Assay, Fluorescence

    64) Product Images from "Glycosylation of the Hemagglutinin Protein of H5N1 Influenza Virus Increases Its Virulence in Mice by Exacerbating the Host Immune Response"

    Article Title: Glycosylation of the Hemagglutinin Protein of H5N1 Influenza Virus Increases Its Virulence in Mice by Exacerbating the Host Immune Response

    Journal: Journal of Virology

    doi: 10.1128/JVI.02215-16

    Expression of viral proteins in infected MDCK cells. MDCK cells were infected with viruses at an MOI of 5. The infected cells were collected at 12 h postinfection, lysed in SDS loading buffer, and further analyzed by Western blotting (A and B). (A) Protein bands of HA, NP, and M1 proteins detected by Western blotting. The intensity of each band was measured by using ImageJ software, and relative intensity ratios of HA, NP, and M1 compared with that of GAPDH were calculated (B). The infected cells were digested with trypsin to obtain a single cell suspension at 12 h postinfection. The samples were stained with rabbit monoclonal antibody to influenza A virus H5N1 HA protein and Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) secondary antibody and then detected on a FACSAria II (BD Biosciences). (C) Mean fluorescence intensity of HA protein was analyzed with FlowJo X 10.0.7r2 (Tree Star, San Carlos, CA). Data are presented as means ± SD ( n = 3). **, P
    Figure Legend Snippet: Expression of viral proteins in infected MDCK cells. MDCK cells were infected with viruses at an MOI of 5. The infected cells were collected at 12 h postinfection, lysed in SDS loading buffer, and further analyzed by Western blotting (A and B). (A) Protein bands of HA, NP, and M1 proteins detected by Western blotting. The intensity of each band was measured by using ImageJ software, and relative intensity ratios of HA, NP, and M1 compared with that of GAPDH were calculated (B). The infected cells were digested with trypsin to obtain a single cell suspension at 12 h postinfection. The samples were stained with rabbit monoclonal antibody to influenza A virus H5N1 HA protein and Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) secondary antibody and then detected on a FACSAria II (BD Biosciences). (C) Mean fluorescence intensity of HA protein was analyzed with FlowJo X 10.0.7r2 (Tree Star, San Carlos, CA). Data are presented as means ± SD ( n = 3). **, P

    Techniques Used: Expressing, Infection, Western Blot, Software, Staining, Fluorescence

    65) Product Images from "Regulation of C3a Receptor Signaling in Human Mast Cells by G Protein Coupled Receptor Kinases"

    Article Title: Regulation of C3a Receptor Signaling in Human Mast Cells by G Protein Coupled Receptor Kinases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0022559

    GRKs do not modulate PMA-induced ERK1/2 phosphorylation. shRNA control, GRK5, or GRK6 KD HMC-1 cells were washed with serum-free medium and exposed to phorol-myristate acetate (PMA, 10 nM) for 0, 5 and 10 min. Cell lysates were separated on SDS-PAGE and blots were probed with anti-phospho-ERK1/2 antibody. The blots were then stripped and reprobed with anti-ERK1/2 antibody followed by anti-rabbit IgG-HRP. Immunoreactive band were visualized by SuperSignal West Femto maximum sensitivity substrate. (A) Representative immunoblots from three similar experiments are shown. (B) ERK1/2 phosphorylation from three experiments was quantified using Image J software.
    Figure Legend Snippet: GRKs do not modulate PMA-induced ERK1/2 phosphorylation. shRNA control, GRK5, or GRK6 KD HMC-1 cells were washed with serum-free medium and exposed to phorol-myristate acetate (PMA, 10 nM) for 0, 5 and 10 min. Cell lysates were separated on SDS-PAGE and blots were probed with anti-phospho-ERK1/2 antibody. The blots were then stripped and reprobed with anti-ERK1/2 antibody followed by anti-rabbit IgG-HRP. Immunoreactive band were visualized by SuperSignal West Femto maximum sensitivity substrate. (A) Representative immunoblots from three similar experiments are shown. (B) ERK1/2 phosphorylation from three experiments was quantified using Image J software.

    Techniques Used: shRNA, SDS Page, Western Blot, Software

    Silencing the expression of GRK2/3 and GRK5/6 enhance C3a-induced ERK1/2 phosphorylation. shRNA control, GRK2, GRK3, GRK5, or GRK6 KD HMC-1 cells were washed with serum-free medium and exposed to C3a (100 nM) for 1, 5 and 10 min. Cell lysates were separated on SDS-PAGE and blots were probed with anti-phospho-ERK1/2 antibody. The blots were then stripped and reprobed with anti-ERK1/2 antibody followed by anti-rabbit IgG-HRP. Immunoreactive band were visualized by SuperSignal West Femto maximum sensitivity substrate. (A, C) Representative immunoblots from three similar experiments are shown. (B, D) ERK1/2 phosphorylation was quantified from 3 experiments using Image J software. Data represent the mean ± SEM from three independent experiments. Statistical significance was determined by unpaired two-tailed t test. *, p
    Figure Legend Snippet: Silencing the expression of GRK2/3 and GRK5/6 enhance C3a-induced ERK1/2 phosphorylation. shRNA control, GRK2, GRK3, GRK5, or GRK6 KD HMC-1 cells were washed with serum-free medium and exposed to C3a (100 nM) for 1, 5 and 10 min. Cell lysates were separated on SDS-PAGE and blots were probed with anti-phospho-ERK1/2 antibody. The blots were then stripped and reprobed with anti-ERK1/2 antibody followed by anti-rabbit IgG-HRP. Immunoreactive band were visualized by SuperSignal West Femto maximum sensitivity substrate. (A, C) Representative immunoblots from three similar experiments are shown. (B, D) ERK1/2 phosphorylation was quantified from 3 experiments using Image J software. Data represent the mean ± SEM from three independent experiments. Statistical significance was determined by unpaired two-tailed t test. *, p

    Techniques Used: Expressing, shRNA, SDS Page, Western Blot, Software, Two Tailed Test

    66) Product Images from "Oral Delivery of a DNA Vaccine Expressing the PrM and E Genes: A Promising Vaccine Strategy against Flavivirus in Ducks"

    Article Title: Oral Delivery of a DNA Vaccine Expressing the PrM and E Genes: A Promising Vaccine Strategy against Flavivirus in Ducks

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-30258-3

    Expression of antigen genes in vitro . ( A ) Genomic structure of DTMUV, which includes genes encoding structural proteins (C, prM and E), nonstructural proteins, 5′-end untranslated region (5′UTR) and 3′UTR. ( B ) Genes encoding the structural proteins prM, E and the signal peptide of C proteins were cloned in to pVAX1 to generate the DNA vaccine plasmid pVAX-SME. (C) COS7 cells were transfected with plasmid pVAX or pVAX-SME. The expression of E protein was detected by indirect immunofluorescence assay using the rabbit anti-DTMUV-E protein polyclonal antibody and the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody. Blue fluorescence indicates nuclei, and green fluorescence indicate DTMUV E proteins. (D ) Duck embryo cells were transfected with plasmid pVAX-SME or pVAX. The expression of prM (left panel) and E (right panel) proteins was checked by Western blotting by using the mouse anti-DTMUV-prM polyclonal antibody combining horseradish peroxidase-conjugated goat anti-mouse antibody, or rabbit anti-DTMUV-E polyclonal antibody combining horseradish peroxidase-conjugated goat anti-rabbit antibody.
    Figure Legend Snippet: Expression of antigen genes in vitro . ( A ) Genomic structure of DTMUV, which includes genes encoding structural proteins (C, prM and E), nonstructural proteins, 5′-end untranslated region (5′UTR) and 3′UTR. ( B ) Genes encoding the structural proteins prM, E and the signal peptide of C proteins were cloned in to pVAX1 to generate the DNA vaccine plasmid pVAX-SME. (C) COS7 cells were transfected with plasmid pVAX or pVAX-SME. The expression of E protein was detected by indirect immunofluorescence assay using the rabbit anti-DTMUV-E protein polyclonal antibody and the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody. Blue fluorescence indicates nuclei, and green fluorescence indicate DTMUV E proteins. (D ) Duck embryo cells were transfected with plasmid pVAX-SME or pVAX. The expression of prM (left panel) and E (right panel) proteins was checked by Western blotting by using the mouse anti-DTMUV-prM polyclonal antibody combining horseradish peroxidase-conjugated goat anti-mouse antibody, or rabbit anti-DTMUV-E polyclonal antibody combining horseradish peroxidase-conjugated goat anti-rabbit antibody.

    Techniques Used: Expressing, In Vitro, Clone Assay, Plasmid Preparation, Transfection, Immunofluorescence, Fluorescence, Western Blot

    67) Product Images from "Tetraspanin CD82 interaction with cholesterol promotes extracellular vesicle–mediated release of ezrin to inhibit tumour cell movement"

    Article Title: Tetraspanin CD82 interaction with cholesterol promotes extracellular vesicle–mediated release of ezrin to inhibit tumour cell movement

    Journal: Journal of Extracellular Vesicles

    doi: 10.1080/20013078.2019.1692417

    CD82 and its cholesterol-binding differentially regulate cellular release of EVs. (a) Extracellular staining by filipin and Alexa488-conjugated CTxb in Du145 transfectants. Equal number of the cells were cultured on glass coverslips for 2 days, then fixed and labelled with filipin or Alexa488-conjugated CTxb. For filipin staining, intercellular regions were imaged. For CTxb staining, pericellular regions were imaged. Scale bar: 10 µm. (b) Distributions of Annexin V and Annexin A2 in Du145 transfectant cells. Alexa488-conjugated recombinant Annexin V was used for phosphatidylserine labelling, while Annexin-A2 Ab was used for Annexin-A2 staining. Scale bar: 10 μm. (c) Colocalization of Ezrin with GM1 or Annexin A2 in EVs. For Ezrin and GM1 co-staining, the cells were labelled with the Abs, Alexa488-conjugated CTxB and DAPI. For Ezrin and Annexin A2 co-staining, the cells were incubated sequentially with the primary Abs, Cy3-conjugated donkey anti-goat IgG, normal goat IgG and Alexa594-conjugated goat anti-mouse IgG. Images were obtained by confocal microscopy. Scale bar: 10 µm. (d) The cells were seeded in six-well plate at 50% confluence and cultured in DMEM containing 1% exosome-depleted FBS for 2 – 3 days. The culture supernatants were collected, spun at 2000 × g for 10 min to remove cell debris, and then analysed with NanoSight instrument for EV number and size. Data are presented as mean ± SD (n = 3 individual experiments). *: p
    Figure Legend Snippet: CD82 and its cholesterol-binding differentially regulate cellular release of EVs. (a) Extracellular staining by filipin and Alexa488-conjugated CTxb in Du145 transfectants. Equal number of the cells were cultured on glass coverslips for 2 days, then fixed and labelled with filipin or Alexa488-conjugated CTxb. For filipin staining, intercellular regions were imaged. For CTxb staining, pericellular regions were imaged. Scale bar: 10 µm. (b) Distributions of Annexin V and Annexin A2 in Du145 transfectant cells. Alexa488-conjugated recombinant Annexin V was used for phosphatidylserine labelling, while Annexin-A2 Ab was used for Annexin-A2 staining. Scale bar: 10 μm. (c) Colocalization of Ezrin with GM1 or Annexin A2 in EVs. For Ezrin and GM1 co-staining, the cells were labelled with the Abs, Alexa488-conjugated CTxB and DAPI. For Ezrin and Annexin A2 co-staining, the cells were incubated sequentially with the primary Abs, Cy3-conjugated donkey anti-goat IgG, normal goat IgG and Alexa594-conjugated goat anti-mouse IgG. Images were obtained by confocal microscopy. Scale bar: 10 µm. (d) The cells were seeded in six-well plate at 50% confluence and cultured in DMEM containing 1% exosome-depleted FBS for 2 – 3 days. The culture supernatants were collected, spun at 2000 × g for 10 min to remove cell debris, and then analysed with NanoSight instrument for EV number and size. Data are presented as mean ± SD (n = 3 individual experiments). *: p

    Techniques Used: Binding Assay, Staining, Cell Culture, Transfection, Recombinant, Incubation, Confocal Microscopy

    68) Product Images from "Activation of Ras-ERK Signaling and GSK-3 by Amyloid Precursor Protein and Amyloid Beta Facilitates Neurodegeneration in Alzheimer’s Disease"

    Article Title: Activation of Ras-ERK Signaling and GSK-3 by Amyloid Precursor Protein and Amyloid Beta Facilitates Neurodegeneration in Alzheimer’s Disease

    Journal: eNeuro

    doi: 10.1523/ENEURO.0149-16.2017

    Analysis of cellular distribution of Ras and P-ERK in neurons treated with oligomeric Aβ42. Primary rat cortical neurons from E18 embryos were cultured for at least 5 d and treated with 2.5 or 5 µ m oligomeric Aβ42 for 24 h. DMSO treatment served as vehicle control. Neurons were immunostained with neuron-specific MAP2 mouse monoclonal antibody and Ras ( A ), P-ERK ( C ), or Cyclin D1 ( E ) rabbit polyclonal antibodies. Arrow points to the magnified image of the cell to show nuclear staining of P-ERK or Cyclin D1. Immunostaining was visualized with Alexa Fluor 594 (red) and 488 (green) fluorophores, respectively. Hoechst staining was used to visualize nuclei (blue). The staining was analyzed with AxioVision Rel 4.8 software for Zeiss microscope. The bar graphs represent the qualitative analyses of the corrected total cell fluorescence for Ras ( n = 30 neurons/group) ( B ), P-ERK ( n = 30 neurons/group) ( D ), and Cyclin D1 ( n = 30 neurons/group) ( F ). Statistical analysis was performed using ANOVA, and the data are representative of three independent experiments. Magnification 63×.
    Figure Legend Snippet: Analysis of cellular distribution of Ras and P-ERK in neurons treated with oligomeric Aβ42. Primary rat cortical neurons from E18 embryos were cultured for at least 5 d and treated with 2.5 or 5 µ m oligomeric Aβ42 for 24 h. DMSO treatment served as vehicle control. Neurons were immunostained with neuron-specific MAP2 mouse monoclonal antibody and Ras ( A ), P-ERK ( C ), or Cyclin D1 ( E ) rabbit polyclonal antibodies. Arrow points to the magnified image of the cell to show nuclear staining of P-ERK or Cyclin D1. Immunostaining was visualized with Alexa Fluor 594 (red) and 488 (green) fluorophores, respectively. Hoechst staining was used to visualize nuclei (blue). The staining was analyzed with AxioVision Rel 4.8 software for Zeiss microscope. The bar graphs represent the qualitative analyses of the corrected total cell fluorescence for Ras ( n = 30 neurons/group) ( B ), P-ERK ( n = 30 neurons/group) ( D ), and Cyclin D1 ( n = 30 neurons/group) ( F ). Statistical analysis was performed using ANOVA, and the data are representative of three independent experiments. Magnification 63×.

    Techniques Used: Cell Culture, Staining, Immunostaining, Software, Microscopy, Fluorescence

    69) Product Images from "Upregulation of glycosaminoglycan synthesis by Neurotropin in nucleus pulposus cells via stimulation of chondroitin sulfate N-acetylgalactosaminyltransferase 1: A new approach to attenuation of intervertebral disc degeneration"

    Article Title: Upregulation of glycosaminoglycan synthesis by Neurotropin in nucleus pulposus cells via stimulation of chondroitin sulfate N-acetylgalactosaminyltransferase 1: A new approach to attenuation of intervertebral disc degeneration

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0202640

    NTP activates AKT phosphorylation. Phosphorylation of AKT by NTP was examined in NP cells by staining with specific antibody against pAKT (Ser473). A) Time courses show the integrated signals of the Alexa Fluor 488-labeled pAKT (shown in green in the photograph) indicated peaks from 15 to 90 minutes after addition of NTP and AsAP. The peak levels of phosphorylation were much higher in the samples co-treated with 0.1, 1.0 and 10 mNU/ml NTP and AsAP than in those treated with AsAP only. Capture of immunofluorescence images and integration of the signal intensity in the cultured cells were performed using the ArrayScan system. Every serial sample was assayed in duplicated wells (Donor, N = 1). Red arrow shows the time point at which the representative photographs were taken. Nuclei are shown in blue by staining with DAPI. Bars are 100 μm. B) Western blot analysis of phosphorylation of AKT using the same antibody. Thirty minutes and 60 minutes after addition of NTP, cells were harvested and whole cell lysate was immunoblotted (N = 2). Addition of 0.1 mNU/ml NTP upregulated phosphorylation of AKT (Ser473) compared with control, but higher concentrations of NTP (1.0 and 10 mNU/ml) were not proportionally more effective (left and right). Addition of AsAP alone was also effective on AKT phosphorylation (right). AKT is constant under all culture conditions.
    Figure Legend Snippet: NTP activates AKT phosphorylation. Phosphorylation of AKT by NTP was examined in NP cells by staining with specific antibody against pAKT (Ser473). A) Time courses show the integrated signals of the Alexa Fluor 488-labeled pAKT (shown in green in the photograph) indicated peaks from 15 to 90 minutes after addition of NTP and AsAP. The peak levels of phosphorylation were much higher in the samples co-treated with 0.1, 1.0 and 10 mNU/ml NTP and AsAP than in those treated with AsAP only. Capture of immunofluorescence images and integration of the signal intensity in the cultured cells were performed using the ArrayScan system. Every serial sample was assayed in duplicated wells (Donor, N = 1). Red arrow shows the time point at which the representative photographs were taken. Nuclei are shown in blue by staining with DAPI. Bars are 100 μm. B) Western blot analysis of phosphorylation of AKT using the same antibody. Thirty minutes and 60 minutes after addition of NTP, cells were harvested and whole cell lysate was immunoblotted (N = 2). Addition of 0.1 mNU/ml NTP upregulated phosphorylation of AKT (Ser473) compared with control, but higher concentrations of NTP (1.0 and 10 mNU/ml) were not proportionally more effective (left and right). Addition of AsAP alone was also effective on AKT phosphorylation (right). AKT is constant under all culture conditions.

    Techniques Used: Staining, Labeling, Immunofluorescence, Cell Culture, Western Blot

    70) Product Images from "Primary rat LSECs preserve their characteristic phenotype after cryopreservation"

    Article Title: Primary rat LSECs preserve their characteristic phenotype after cryopreservation

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-32103-z

    Expression of main endocytosis receptors by fLSEC and cLSECs. In all micrographs, the fLSECs are shown in the left panel and cLSECs in the right panel. The cultures were fixed with paraformaldehyde, permeabilized with Triton X, and immune labeled with antibodies against stabilin-2 ( a and b ), mannose receptor (MR) ( d and e ), and FcγRIIb2 ( g and h ). Positive immunolabeling was visualized with Alexa Fluor-488 secondary antibodies (green fluorescence). Cell nuclei were stained with DAPI (blue fluorescence). ( g — i ) The average fluorescence intensity per cell for each receptor protein was measured and the results expressed as relative expression, where the expression of the different markers in fLSECs equals 1. The p value is shown, which was calculated using the Excel two-tailed paired t -test assuming unequal variation. Statistical details are presented in Table 2 under Materials and Methods.
    Figure Legend Snippet: Expression of main endocytosis receptors by fLSEC and cLSECs. In all micrographs, the fLSECs are shown in the left panel and cLSECs in the right panel. The cultures were fixed with paraformaldehyde, permeabilized with Triton X, and immune labeled with antibodies against stabilin-2 ( a and b ), mannose receptor (MR) ( d and e ), and FcγRIIb2 ( g and h ). Positive immunolabeling was visualized with Alexa Fluor-488 secondary antibodies (green fluorescence). Cell nuclei were stained with DAPI (blue fluorescence). ( g — i ) The average fluorescence intensity per cell for each receptor protein was measured and the results expressed as relative expression, where the expression of the different markers in fLSECs equals 1. The p value is shown, which was calculated using the Excel two-tailed paired t -test assuming unequal variation. Statistical details are presented in Table 2 under Materials and Methods.

    Techniques Used: Expressing, Labeling, Immunolabeling, Fluorescence, Staining, Two Tailed Test

    71) Product Images from "Differential susceptibility of bovine caruncular and trophoblast cell lines to infection with high and low virulence isolates of Neospora caninum"

    Article Title: Differential susceptibility of bovine caruncular and trophoblast cell lines to infection with high and low virulence isolates of Neospora caninum

    Journal: Parasites & Vectors

    doi: 10.1186/s13071-017-2409-9

    Adhesion assay in F3 and BCEC-1 cells infected by Nc-Spain7 and Nc-Spain1H at 4 hpi. Double immunofluorescence staining was performed, and adhered extracellular tachyzoites were stained with Alexa Fluor® 488 ( green ) and Alexa Fluor® 594 ( red ), whereas intracellular tachyzoites were stained with Alexa Fluor® 594 ( red) . Nuclei were stained with DAPI ( blue ). Tachyzoites were counted in 10 arbitrarily selected fields, and the percentage of intracellular tachyzoites relative to the number of total adhered tachyzoites at 4 hpi was calculated. Representative images at a magnification of 1000× ( a ) show the adhesion assay performed in F3 and BCEC-1 cells infected with both isolates. The graph ( b ) represents the percentage of intracellular tachyzoites of Nc-Spain7 and Nc-Spain1H relative to the total number of intra- and extracellular tachyzoites adhered to F3 and BCEC-1 cells. Each column and error bar represents the mean and the SD of 4 replicates from 2 independent assays. BCEC-1 cells showed a significantly higher percentage of intracellular tachyzoites than F3 cells ( P
    Figure Legend Snippet: Adhesion assay in F3 and BCEC-1 cells infected by Nc-Spain7 and Nc-Spain1H at 4 hpi. Double immunofluorescence staining was performed, and adhered extracellular tachyzoites were stained with Alexa Fluor® 488 ( green ) and Alexa Fluor® 594 ( red ), whereas intracellular tachyzoites were stained with Alexa Fluor® 594 ( red) . Nuclei were stained with DAPI ( blue ). Tachyzoites were counted in 10 arbitrarily selected fields, and the percentage of intracellular tachyzoites relative to the number of total adhered tachyzoites at 4 hpi was calculated. Representative images at a magnification of 1000× ( a ) show the adhesion assay performed in F3 and BCEC-1 cells infected with both isolates. The graph ( b ) represents the percentage of intracellular tachyzoites of Nc-Spain7 and Nc-Spain1H relative to the total number of intra- and extracellular tachyzoites adhered to F3 and BCEC-1 cells. Each column and error bar represents the mean and the SD of 4 replicates from 2 independent assays. BCEC-1 cells showed a significantly higher percentage of intracellular tachyzoites than F3 cells ( P

    Techniques Used: Cell Adhesion Assay, Infection, Double Immunofluorescence Staining, Staining

    72) Product Images from "Interleukin-like EMT inducer (ILEI) promotes melanoma invasiveness and is transcriptionally up-regulated by upstream stimulatory factor-1 (USF-1)"

    Article Title: Interleukin-like EMT inducer (ILEI) promotes melanoma invasiveness and is transcriptionally up-regulated by upstream stimulatory factor-1 (USF-1)

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.003616

    USF-1 interacts with the ILEI promoter sequence. A, 5′-biotin–tagged ILEI promoter oligonucleotide constructs with WT or mutant E-box. Biotin pulldown analysis of nuclear extracts from 501-Mel melanoma cells, followed by immunoblot for USF-1. B, PCR primers flanking the ILEI promoter E-box used in ChIP analysis. ChIP analysis of 501-Mel melanoma cell lines immunoprecipitated with control IgG or USF-1 antibody. PCR analysis conducted with primers targeting FAM3C, TYR, or HO1 promoter.
    Figure Legend Snippet: USF-1 interacts with the ILEI promoter sequence. A, 5′-biotin–tagged ILEI promoter oligonucleotide constructs with WT or mutant E-box. Biotin pulldown analysis of nuclear extracts from 501-Mel melanoma cells, followed by immunoblot for USF-1. B, PCR primers flanking the ILEI promoter E-box used in ChIP analysis. ChIP analysis of 501-Mel melanoma cell lines immunoprecipitated with control IgG or USF-1 antibody. PCR analysis conducted with primers targeting FAM3C, TYR, or HO1 promoter.

    Techniques Used: Sequencing, Construct, Mutagenesis, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Immunoprecipitation

    73) Product Images from "Peculiar Expression of CD3-Epsilon in Kidney of Ginbuna Crucian Carp"

    Article Title: Peculiar Expression of CD3-Epsilon in Kidney of Ginbuna Crucian Carp

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01321

    Immuno-histochemical analysis of anti-CD3ε-positive cells in tissues. Cryostat sections were stained with anti-gCD3ε Ab (A = Thymus, B = Gill, C = Trunk-Kidney, D = Spleen, and E = Intestine) or isotype Ab (a = Thymus, b = Gill, c = Trunk-Kidney, d = Spleen, and e = Intestine) followed by Alexa Fluor ® 488 goat anti-rabbit IgG (green). Nuclei were stained with DAPI (blue). Scale bar = 20 µm (A–E) . Antibody positive cells (green) were analyzed in high magnification (F) . Antibody positive cell (green) were merged with nuclei staining (blue) (G) . Scale bar = 1 µm (F,G) .
    Figure Legend Snippet: Immuno-histochemical analysis of anti-CD3ε-positive cells in tissues. Cryostat sections were stained with anti-gCD3ε Ab (A = Thymus, B = Gill, C = Trunk-Kidney, D = Spleen, and E = Intestine) or isotype Ab (a = Thymus, b = Gill, c = Trunk-Kidney, d = Spleen, and e = Intestine) followed by Alexa Fluor ® 488 goat anti-rabbit IgG (green). Nuclei were stained with DAPI (blue). Scale bar = 20 µm (A–E) . Antibody positive cells (green) were analyzed in high magnification (F) . Antibody positive cell (green) were merged with nuclei staining (blue) (G) . Scale bar = 1 µm (F,G) .

    Techniques Used: Staining

    Dual fluorescence analysis of CD3ε + , Zap-70 with lymphocyte markers in tissues. Leukocytes from spleen, kidney, and thymus were stained with the anti-CD4-1 and CD8α mAbs followed by Alexa Fluor ® 488 anti-rat IgG, and stained with anti-gCD3ε Ab or anti-hZap-70 mAb followed by 647 goat anti-rabbit IgG. (A–C) Lymphocytes were gated on FS and SS dot plot. (D) Leukocytes from peripheral blood leukocytes were stained with anti-CD4-1, CD8α, IgM, phagocyte, and thrombocyte mAbs, respectively, followed by Alexa Fluor ® 488 goat anti-rat or mouse IgG, and stained with anti-gCD3ε Ab followed by Alexa Fluor ® 647 goat anti-rabbit IgG. Mean ± SD of more than three independent experiments are shown.
    Figure Legend Snippet: Dual fluorescence analysis of CD3ε + , Zap-70 with lymphocyte markers in tissues. Leukocytes from spleen, kidney, and thymus were stained with the anti-CD4-1 and CD8α mAbs followed by Alexa Fluor ® 488 anti-rat IgG, and stained with anti-gCD3ε Ab or anti-hZap-70 mAb followed by 647 goat anti-rabbit IgG. (A–C) Lymphocytes were gated on FS and SS dot plot. (D) Leukocytes from peripheral blood leukocytes were stained with anti-CD4-1, CD8α, IgM, phagocyte, and thrombocyte mAbs, respectively, followed by Alexa Fluor ® 488 goat anti-rat or mouse IgG, and stained with anti-gCD3ε Ab followed by Alexa Fluor ® 647 goat anti-rabbit IgG. Mean ± SD of more than three independent experiments are shown.

    Techniques Used: Fluorescence, Staining

    Percentages of gCD3ε-positive cells in the lymphocyte fraction of tissues. Leukocytes from thymus, spleen, trunk-kidney, gill intestine, and peripheral blood leukocytes were stained with anti-gCD3ε Ab followed by Alexa Fluor ® 647 goat anti-rabbit IgG. Lymphocytes were gated on FS and SS dot plot. Dotted lines show negative control stained with isotype antibody, and black solid lines with gray shadow show anti-gCD3ε Ab-positive cells. Each figure is representative of more than seven analyses with mean value and SD (mean ± SD).
    Figure Legend Snippet: Percentages of gCD3ε-positive cells in the lymphocyte fraction of tissues. Leukocytes from thymus, spleen, trunk-kidney, gill intestine, and peripheral blood leukocytes were stained with anti-gCD3ε Ab followed by Alexa Fluor ® 647 goat anti-rabbit IgG. Lymphocytes were gated on FS and SS dot plot. Dotted lines show negative control stained with isotype antibody, and black solid lines with gray shadow show anti-gCD3ε Ab-positive cells. Each figure is representative of more than seven analyses with mean value and SD (mean ± SD).

    Techniques Used: Staining, Negative Control

    74) Product Images from "BgTEP: An Antiprotease Involved in Innate Immune Sensing in Biomphalaria glabrata"

    Article Title: BgTEP: An Antiprotease Involved in Innate Immune Sensing in Biomphalaria glabrata

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01206

    BgTEP-positive cells do not phagocyte. Phagocytosis of green fluorescent zymosan particles in hemocytes observed by confocal microscopy. On picture 1, Alexa-594 phalloidin was used to label actin of all hemocytes (red). Phagocytosis assay was monitored using green fluorescent yeasts. On picture 2, BgTEP-positive cells were detected by immunolocalization using the antibody anti-BgTEP-PEP and an Alexa Fluor 594 dye conjugated to the secondary antibody (red).
    Figure Legend Snippet: BgTEP-positive cells do not phagocyte. Phagocytosis of green fluorescent zymosan particles in hemocytes observed by confocal microscopy. On picture 1, Alexa-594 phalloidin was used to label actin of all hemocytes (red). Phagocytosis assay was monitored using green fluorescent yeasts. On picture 2, BgTEP-positive cells were detected by immunolocalization using the antibody anti-BgTEP-PEP and an Alexa Fluor 594 dye conjugated to the secondary antibody (red).

    Techniques Used: Confocal Microscopy, Phagocytosis Assay

    75) Product Images from "Successful development of squamous cell carcinoma and hyperplasia in RGEN-mediated p27 KO mice after the treatment of DMBA and TPA"

    Article Title: Successful development of squamous cell carcinoma and hyperplasia in RGEN-mediated p27 KO mice after the treatment of DMBA and TPA

    Journal: Laboratory Animal Research

    doi: 10.5625/lar.2018.34.3.118

    Expression analysis of cell-cycle regulator proteins. An alteration in the expressions of Cyclin D1, CDK2 and CDK4 proteins were determined in DT-treated p27 (IΔ) KO mice by Western blot assays using HRP-labeled anti-rabbit IgG antibody. Band intensities were determined using an imaging densitometer, and the expression level of 6 proteins were evaluated relative to the intensity of actin bands. The data represents the means±SD (n=8). *, indicates P
    Figure Legend Snippet: Expression analysis of cell-cycle regulator proteins. An alteration in the expressions of Cyclin D1, CDK2 and CDK4 proteins were determined in DT-treated p27 (IΔ) KO mice by Western blot assays using HRP-labeled anti-rabbit IgG antibody. Band intensities were determined using an imaging densitometer, and the expression level of 6 proteins were evaluated relative to the intensity of actin bands. The data represents the means±SD (n=8). *, indicates P

    Techniques Used: Expressing, Mouse Assay, Western Blot, Labeling, Imaging

    Expression analysis of tumor suppressor protein. An alteration in the expressions of p53 and p27 proteins were determined in DT-treated p27 (IΔ) KO mice by Western blot assays using HRP-labeled anti-rabbit IgG antibody. Band intensities were determined using an imaging densitometer, and the expression level of 6 proteins were evaluated relative to the intensity of actin bands. The data represents the means±SD (n=8). *, indicates P
    Figure Legend Snippet: Expression analysis of tumor suppressor protein. An alteration in the expressions of p53 and p27 proteins were determined in DT-treated p27 (IΔ) KO mice by Western blot assays using HRP-labeled anti-rabbit IgG antibody. Band intensities were determined using an imaging densitometer, and the expression level of 6 proteins were evaluated relative to the intensity of actin bands. The data represents the means±SD (n=8). *, indicates P

    Techniques Used: Expressing, Mouse Assay, Western Blot, Labeling, Imaging

    Expression analysis of apoptotic proteins. Alterations in the expressions of Bax, Bcl-2 and Caspase-3 proteins were determined in DT-treated p27 (IΔ) KO mice by Western blot assays using HRP-labeled anti-rabbit IgG antibody. Band intensities were determined using an imaging densitometer, and the expression level of 6 proteins were evaluated relative to the intensity of actin bands. The data represents the means±SD (n=8). *, indicates P
    Figure Legend Snippet: Expression analysis of apoptotic proteins. Alterations in the expressions of Bax, Bcl-2 and Caspase-3 proteins were determined in DT-treated p27 (IΔ) KO mice by Western blot assays using HRP-labeled anti-rabbit IgG antibody. Band intensities were determined using an imaging densitometer, and the expression level of 6 proteins were evaluated relative to the intensity of actin bands. The data represents the means±SD (n=8). *, indicates P

    Techniques Used: Expressing, Mouse Assay, Western Blot, Labeling, Imaging

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    Blocking Assay:

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    Incubation:

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    Cell Culture:

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    SDS Page:

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    Imaging:

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    Inverted Microscopy:

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    Protein Concentration:

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    Immunofluorescence:

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    Article Snippet: Paragraph title: Immunofluorescence analysis ... The cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody (1:100; cat. no. A-11034; Invitrogen) at 37°C for 1 h and stained with 4,6-diamino-2-phenyl indole at 37°C for 5 min to visualize the nuclei.

    Article Title: Phosphatidylserine Exposure Controls Viral Innate Immune Responses by Microglia
    Article Snippet: Paragraph title: Immunofluorescence ... Secondary antibodies (1:100) included Alexa Fluor 488 goat anti-rabbit (Thermo Fisher Scientific Cat. #A-11034; RRID: AB_2576217), Alexa Fluor 633 goat anti-rat (Thermo Fisher Scientific Cat. #A-21094; RRID: AB_2535749), and Alexa Fluor 633 goat anti-mouse (Thermo Fisher Scientific Cat. #A-21052; RRID: AB_2535719).

    Article Title: Nuclear Receptor Nr4a1 Regulates Striatal Striosome Development and Dopamine D1 Receptor Signaling
    Article Snippet: Paragraph title: Tissue preparation and immunofluorescence ... The respective secondary antibodies used were as follows: anti-mouse Alexa Fluor 488 (1:400; catalog #A-11008, Thermo Fisher Scientific), anti-mouse Alexa Fluor 594 (1:400; catalog #A-11005, Thermo Fisher Scientific), anti-rabbit Alexa Fluor 488 (1:400; catalog #A-11034, Thermo Fisher Scientific), or anti-rabbit Alexa Fluor 594 (1:400; catalog #A-11012, Thermo Fisher Scientific).

    Article Title: Salidroside Inhibits Myogenesis by Modulating p-Smad3-Induced Myf5 Transcription
    Article Snippet: Paragraph title: Immunoblotting and Immunofluorescence Assay ... After that, the cells were incubated with a primary antibody against E-MHC (Hybridoma Bank, BF-G6), p-Smad3, Myf5 or myogenin at 4°C overnight (1:200 dilutions), followed by incubation with the Alexa Fluor 594 (Invitrogen, A-11032) fluorescent dye conjugated to an anti-mouse secondary antibody or Alexa Fluor 488 (Invitrogen, A-11034) fluorescent dye conjugated to an anti-rabbit secondary antibody.

    Fluorescence:

    Article Title: miR-127 aggravates myocardial failure by promoting the TGF-β1/Smad3 signaling
    Article Snippet: The cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody (1:100; cat. no. A-11034; Invitrogen) at 37°C for 1 h and stained with 4,6-diamino-2-phenyl indole at 37°C for 5 min to visualize the nuclei. .. The cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody (1:100; cat. no. A-11034; Invitrogen) at 37°C for 1 h and stained with 4,6-diamino-2-phenyl indole at 37°C for 5 min to visualize the nuclei.

    Article Title: Chromatin swelling drives neutrophil extracellular trap release
    Article Snippet: Subsequently cells were stained with monoclonal anti-human MPO (IgG, mouse, 1:500) (ab25989, Abcam), monoclonal anti-human α-Tubulin (IgG, rabbit, 1:50) (#2125, Cell Signaling Technology) or polyclonal anti-human lamin B1 (IgG, rabbit, 1:1000) (ab16048, Abcam) as primary antibodies over night (4 °C) and visualized with polyclonal anti-mouse Alexa488 (IgG, goat, 1:1000) (#4408, Cell Signaling Technology) or polyclonal anti-rabbit Alexa488 (IgG, goat, 1:500) (A-11034, ThermoFisher Scientific) as secondary antibodies. .. After complete drying and fixation with nail polish, samples were imaged with 40x magnification (Plan-Neofluar 40×/1.30 oil Iris/4440456-0000-000, Zeiss) in a fluorescence microscope (AxioImager M1, Software: AxioVision Rel.4.7, Zeiss) or ×60 magnified with confocal laser scanning microscopy (Olympus IX83 inverted microscope, software: Olympus Fluoview v.4.2).

    Article Title: PAX6 does not regulate Nfia and Nfib expression during neocortical development
    Article Snippet: Chromogenic immunohistochemistry (IHC) or fluorescence immunohistochemistry (IF) was conducted as previously described with minor modifications . .. For IF, Alexa Fluor 488-conjugated goat anti-chicken (1:500; A-11039, Invitrogen) and Alexa Fluor 555-conjugated goat anti-rabbit (1:500; A-11034, Invitrogen) secondary antibodies were used for detection.

    Article Title: Salidroside Inhibits Myogenesis by Modulating p-Smad3-Induced Myf5 Transcription
    Article Snippet: After that, the cells were incubated with a primary antibody against E-MHC (Hybridoma Bank, BF-G6), p-Smad3, Myf5 or myogenin at 4°C overnight (1:200 dilutions), followed by incubation with the Alexa Fluor 594 (Invitrogen, A-11032) fluorescent dye conjugated to an anti-mouse secondary antibody or Alexa Fluor 488 (Invitrogen, A-11034) fluorescent dye conjugated to an anti-rabbit secondary antibody. .. The fluorescence areas, the number of fluorescence-positive nuclei and total nuclei with DAPI staining were counted with Image-Pro plus 6.0 (Media Cybernetics, Inc., United States).

    Isolation:

    Article Title: Chromatin swelling drives neutrophil extracellular trap release
    Article Snippet: Staining procedures Fresh isolated human neutrophils (200,000 per well) were seeded on pretreated (99% alcohol) glass cover slips (#1.5) in 24-well plates (Greiner bio-one) and NET formation induced with 100 nM PMA. .. Subsequently cells were stained with monoclonal anti-human MPO (IgG, mouse, 1:500) (ab25989, Abcam), monoclonal anti-human α-Tubulin (IgG, rabbit, 1:50) (#2125, Cell Signaling Technology) or polyclonal anti-human lamin B1 (IgG, rabbit, 1:1000) (ab16048, Abcam) as primary antibodies over night (4 °C) and visualized with polyclonal anti-mouse Alexa488 (IgG, goat, 1:1000) (#4408, Cell Signaling Technology) or polyclonal anti-rabbit Alexa488 (IgG, goat, 1:500) (A-11034, ThermoFisher Scientific) as secondary antibodies.

    Microscopy:

    Article Title: High-efficiency RNA-based reprogramming of human primary fibroblasts
    Article Snippet: Secondary antibodies used were: Alexa Fluor 594 donkey anti-goat IgG (H + L) (A-11058) 1:250, Alexa Fluor 594 goat anti-mouse IgG (H + L) (A-11005) 1:250, Alexa Fluor 594 goat anti-rat IgG (H + L) (A-11007), Alexa Fluor 594 goat anti-rabbit IgG (H + L) (A-11037), and Alexa Fluor 488 goat anti-rabbit IgG (H + L) (A-11034), all from Thermo Fischer Scientific. .. Images were acquired using a Nikon Eclipse 90i upright microscope using a 10× objective.

    Article Title: miR-127 aggravates myocardial failure by promoting the TGF-β1/Smad3 signaling
    Article Snippet: The cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody (1:100; cat. no. A-11034; Invitrogen) at 37°C for 1 h and stained with 4,6-diamino-2-phenyl indole at 37°C for 5 min to visualize the nuclei. .. The cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody (1:100; cat. no. A-11034; Invitrogen) at 37°C for 1 h and stained with 4,6-diamino-2-phenyl indole at 37°C for 5 min to visualize the nuclei.

    Article Title: Light-sheet microscopy imaging of a whole cleared rat brain with Thy1-GFP transgene
    Article Snippet: GFP was detected using rabbit primary antiGFP antibody (#MB598, MBL) followed by incubation with secondary anti-rabbit antibody conjugated with AlexaFluor488 (#A-11034, ThermoFisher Scientific). .. Confocal imaging was performed using LSM780 microscope (Zeiss).

    Article Title: Chromatin swelling drives neutrophil extracellular trap release
    Article Snippet: Subsequently cells were stained with monoclonal anti-human MPO (IgG, mouse, 1:500) (ab25989, Abcam), monoclonal anti-human α-Tubulin (IgG, rabbit, 1:50) (#2125, Cell Signaling Technology) or polyclonal anti-human lamin B1 (IgG, rabbit, 1:1000) (ab16048, Abcam) as primary antibodies over night (4 °C) and visualized with polyclonal anti-mouse Alexa488 (IgG, goat, 1:1000) (#4408, Cell Signaling Technology) or polyclonal anti-rabbit Alexa488 (IgG, goat, 1:500) (A-11034, ThermoFisher Scientific) as secondary antibodies. .. Then, chromatin was stained with Hoechst if applicable and cover slips were mounted with Faramount Mounting Medium (Dako Agilent Technologies) on microscopy slide.

    Article Title: Nuclear Receptor Nr4a1 Regulates Striatal Striosome Development and Dopamine D1 Receptor Signaling
    Article Snippet: Serial coronal section (16 µm) were cut on a Leica cryostat, collected on Superfrost Plus Microscope Slides (Thermo Fisher Scientific), and frozen at −20°C. .. The respective secondary antibodies used were as follows: anti-mouse Alexa Fluor 488 (1:400; catalog #A-11008, Thermo Fisher Scientific), anti-mouse Alexa Fluor 594 (1:400; catalog #A-11005, Thermo Fisher Scientific), anti-rabbit Alexa Fluor 488 (1:400; catalog #A-11034, Thermo Fisher Scientific), or anti-rabbit Alexa Fluor 594 (1:400; catalog #A-11012, Thermo Fisher Scientific).

    Article Title: Salidroside Inhibits Myogenesis by Modulating p-Smad3-Induced Myf5 Transcription
    Article Snippet: After that, the cells were incubated with a primary antibody against E-MHC (Hybridoma Bank, BF-G6), p-Smad3, Myf5 or myogenin at 4°C overnight (1:200 dilutions), followed by incubation with the Alexa Fluor 594 (Invitrogen, A-11032) fluorescent dye conjugated to an anti-mouse secondary antibody or Alexa Fluor 488 (Invitrogen, A-11034) fluorescent dye conjugated to an anti-rabbit secondary antibody. .. Photo capture was performed using a Nikon laser microscope (Eclipse E600, Nikon Instruments, Inc., Japan).

    Mouse Assay:

    Article Title: Nuclear Receptor Nr4a1 Regulates Striatal Striosome Development and Dopamine D1 Receptor Signaling
    Article Snippet: Postnatal day 3 (P3) mice were rapidly killed by decapitation, and brains were removed, washed in ice-cold PBS, and postfixed for 24 h at 4°C in 4% PFA. .. The respective secondary antibodies used were as follows: anti-mouse Alexa Fluor 488 (1:400; catalog #A-11008, Thermo Fisher Scientific), anti-mouse Alexa Fluor 594 (1:400; catalog #A-11005, Thermo Fisher Scientific), anti-rabbit Alexa Fluor 488 (1:400; catalog #A-11034, Thermo Fisher Scientific), or anti-rabbit Alexa Fluor 594 (1:400; catalog #A-11012, Thermo Fisher Scientific).

    Immunostaining:

    Article Title: Administration of Oxygen Ultra-Fine Bubbles Improves Nerve Dysfunction in a Rat Sciatic Nerve Crush Injury Model
    Article Snippet: Paragraph title: 4.7. Immunostaining of Sciatic Nerves ... After blocking with PBS containing 0.2% Triton X and 5% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA), they were incubated with primary antibodies against MBP (mouse; 1:1000; NE1018; Calbiochem, San Diego, CA, USA) and NF200 (rabbit; 1:1000; N4142; Sigma-Aldrich) overnight at 4 °C inside a humidified chamber, followed by incubation with the appropriate secondary antibodies including Alexa Fluor 488 goat anti-rabbit IgG (1:1000; A-11034; Molecular Probes, Eugene, OR, USA) and Alexa Fluor 568 goat anti-mouse IgG (1:1000; A-11004; Molecular Probes).

    Bradford Protein Assay:

    Article Title: Salidroside Inhibits Myogenesis by Modulating p-Smad3-Induced Myf5 Transcription
    Article Snippet: Supernatants were collected and the protein concentration was determined using the Bradford protein assay reagent (Bio-Rad, 500-0203). .. After that, the cells were incubated with a primary antibody against E-MHC (Hybridoma Bank, BF-G6), p-Smad3, Myf5 or myogenin at 4°C overnight (1:200 dilutions), followed by incubation with the Alexa Fluor 594 (Invitrogen, A-11032) fluorescent dye conjugated to an anti-mouse secondary antibody or Alexa Fluor 488 (Invitrogen, A-11034) fluorescent dye conjugated to an anti-rabbit secondary antibody.

    In Situ Hybridization:

    Article Title: Intracellular uptake of macromolecules by brain lymphatic endothelial cells during zebrafish embryonic development
    Article Snippet: Paragraph title: Whole-mount in situ hybridization ... Secondary fluorescent antibodies goat α–Chicken IgG 488 (1:200, Lifetech, #A-11039) and goat α-Rabbit 488 (1:200, Lifetech, #A-11034) were both applied overnight at 4°C.

    Plasmid Preparation:

    Article Title: High-efficiency RNA-based reprogramming of human primary fibroblasts
    Article Snippet: Secondary antibodies used were: Alexa Fluor 594 donkey anti-goat IgG (H + L) (A-11058) 1:250, Alexa Fluor 594 goat anti-mouse IgG (H + L) (A-11005) 1:250, Alexa Fluor 594 goat anti-rat IgG (H + L) (A-11007), Alexa Fluor 594 goat anti-rabbit IgG (H + L) (A-11037), and Alexa Fluor 488 goat anti-rabbit IgG (H + L) (A-11034), all from Thermo Fischer Scientific. .. After staining, slides were mounted in mounting medium with DAPI (4',6-diamidino-2-phenylindole) (Vector Laboratories).

    Article Title: Nuclear Receptor Nr4a1 Regulates Striatal Striosome Development and Dopamine D1 Receptor Signaling
    Article Snippet: The respective secondary antibodies used were as follows: anti-mouse Alexa Fluor 488 (1:400; catalog #A-11008, Thermo Fisher Scientific), anti-mouse Alexa Fluor 594 (1:400; catalog #A-11005, Thermo Fisher Scientific), anti-rabbit Alexa Fluor 488 (1:400; catalog #A-11034, Thermo Fisher Scientific), or anti-rabbit Alexa Fluor 594 (1:400; catalog #A-11012, Thermo Fisher Scientific). .. The respective secondary antibodies used were as follows: anti-mouse Alexa Fluor 488 (1:400; catalog #A-11008, Thermo Fisher Scientific), anti-mouse Alexa Fluor 594 (1:400; catalog #A-11005, Thermo Fisher Scientific), anti-rabbit Alexa Fluor 488 (1:400; catalog #A-11034, Thermo Fisher Scientific), or anti-rabbit Alexa Fluor 594 (1:400; catalog #A-11012, Thermo Fisher Scientific).

    Software:

    Article Title: Chromatin swelling drives neutrophil extracellular trap release
    Article Snippet: Subsequently cells were stained with monoclonal anti-human MPO (IgG, mouse, 1:500) (ab25989, Abcam), monoclonal anti-human α-Tubulin (IgG, rabbit, 1:50) (#2125, Cell Signaling Technology) or polyclonal anti-human lamin B1 (IgG, rabbit, 1:1000) (ab16048, Abcam) as primary antibodies over night (4 °C) and visualized with polyclonal anti-mouse Alexa488 (IgG, goat, 1:1000) (#4408, Cell Signaling Technology) or polyclonal anti-rabbit Alexa488 (IgG, goat, 1:500) (A-11034, ThermoFisher Scientific) as secondary antibodies. .. After complete drying and fixation with nail polish, samples were imaged with 40x magnification (Plan-Neofluar 40×/1.30 oil Iris/4440456-0000-000, Zeiss) in a fluorescence microscope (AxioImager M1, Software: AxioVision Rel.4.7, Zeiss) or ×60 magnified with confocal laser scanning microscopy (Olympus IX83 inverted microscope, software: Olympus Fluoview v.4.2).

    Article Title: Administration of Oxygen Ultra-Fine Bubbles Improves Nerve Dysfunction in a Rat Sciatic Nerve Crush Injury Model
    Article Snippet: After blocking with PBS containing 0.2% Triton X and 5% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA), they were incubated with primary antibodies against MBP (mouse; 1:1000; NE1018; Calbiochem, San Diego, CA, USA) and NF200 (rabbit; 1:1000; N4142; Sigma-Aldrich) overnight at 4 °C inside a humidified chamber, followed by incubation with the appropriate secondary antibodies including Alexa Fluor 488 goat anti-rabbit IgG (1:1000; A-11034; Molecular Probes, Eugene, OR, USA) and Alexa Fluor 568 goat anti-mouse IgG (1:1000; A-11004; Molecular Probes). .. The myelinated ratio was calculated as the number of both MBP- and NF200-positive axons (myelinated axons) to the number of NF-200 positive axons (total axons) using NIS Elements BR 3.00, SP3 software (Laboratory Imaging, Nikon, Tokyo, Japan).

    Negative Control:

    Article Title: miR-127 aggravates myocardial failure by promoting the TGF-β1/Smad3 signaling
    Article Snippet: PBS instead of the primary antibody was used as a negative control. .. The cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody (1:100; cat. no. A-11034; Invitrogen) at 37°C for 1 h and stained with 4,6-diamino-2-phenyl indole at 37°C for 5 min to visualize the nuclei.

    Next-Generation Sequencing:

    Article Title: Intracellular uptake of macromolecules by brain lymphatic endothelial cells during zebrafish embryonic development
    Article Snippet: After stringency washes, 10% NGS/PBS (Block) was added for 1 hr at room temperature, followed with anti-dig POD antibody (1:1000, Roche #11207733910 TSA Cyanine 3 Tyramide Reagent Pack) added in Block overnight at 4°C. .. Secondary fluorescent antibodies goat α–Chicken IgG 488 (1:200, Lifetech, #A-11039) and goat α-Rabbit 488 (1:200, Lifetech, #A-11034) were both applied overnight at 4°C.

    Marker:

    Article Title: High-efficiency RNA-based reprogramming of human primary fibroblasts
    Article Snippet: In the analysis of teratomas, the H & E staining and single-staining immunofluorescence analysis of expression of each germline-specific marker (TUJ1, vimentin, and Endo-A) were performed on separate, consecutively cut sections. .. Secondary antibodies used were: Alexa Fluor 594 donkey anti-goat IgG (H + L) (A-11058) 1:250, Alexa Fluor 594 goat anti-mouse IgG (H + L) (A-11005) 1:250, Alexa Fluor 594 goat anti-rat IgG (H + L) (A-11007), Alexa Fluor 594 goat anti-rabbit IgG (H + L) (A-11037), and Alexa Fluor 488 goat anti-rabbit IgG (H + L) (A-11034), all from Thermo Fischer Scientific.

    Staining:

    Article Title: Platelet Lysate-Derived Neuropeptide y Influences Migration and Angiogenesis of Human Adipose Tissue-Derived Stromal Cells
    Article Snippet: Goat Anti-Rabbit IgG (H + L) (Highly Cross-Adsorbed Secondary antibodies, Alexa Fluor-488, Thermo Fisher, Cat. No. #A-11034) for 45 minutes at room temperature in the dark were added after washing in PBS. .. Nuclei were stained with DAPI (4′−6′-Diamidino-2-phenylindole, powder ≥98%; Sigma, Milan, Italy Cat. N. D9542).

    Article Title: High-efficiency RNA-based reprogramming of human primary fibroblasts
    Article Snippet: Paragraph title: Immunofluorescence analysis and H & E staining ... Secondary antibodies used were: Alexa Fluor 594 donkey anti-goat IgG (H + L) (A-11058) 1:250, Alexa Fluor 594 goat anti-mouse IgG (H + L) (A-11005) 1:250, Alexa Fluor 594 goat anti-rat IgG (H + L) (A-11007), Alexa Fluor 594 goat anti-rabbit IgG (H + L) (A-11037), and Alexa Fluor 488 goat anti-rabbit IgG (H + L) (A-11034), all from Thermo Fischer Scientific.

    Article Title: Rotation of stress fibers as a single wheel in migrating fish keratocytes
    Article Snippet: Paragraph title: Fixed cell staining ... The cells were then incubated with primary antibody: rabbit polyclonal myosin IIA (1:200 dilution, M8064, Sigma-Aldrich) and Alexa Fluor 546 phalloidin (0.33 units/ml, A22283; Life Technologies, Carlsbad, CA) for 60 min. After several washes with 0.2% gelatin, the cells were incubated with secondary antibody: Alexa Fluor 488 Anti-rabbit IgG (1:2,000 dilution, A-11034, Life Technologies) for 60 min.

    Article Title: miR-127 aggravates myocardial failure by promoting the TGF-β1/Smad3 signaling
    Article Snippet: .. The cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody (1:100; cat. no. A-11034; Invitrogen) at 37°C for 1 h and stained with 4,6-diamino-2-phenyl indole at 37°C for 5 min to visualize the nuclei. .. The images were captured using a fluorescence microscope (model DMI-4000B; Leica Microsystems GmbH, Wetzlar, Germany).

    Article Title: Chromatin swelling drives neutrophil extracellular trap release
    Article Snippet: .. Subsequently cells were stained with monoclonal anti-human MPO (IgG, mouse, 1:500) (ab25989, Abcam), monoclonal anti-human α-Tubulin (IgG, rabbit, 1:50) (#2125, Cell Signaling Technology) or polyclonal anti-human lamin B1 (IgG, rabbit, 1:1000) (ab16048, Abcam) as primary antibodies over night (4 °C) and visualized with polyclonal anti-mouse Alexa488 (IgG, goat, 1:1000) (#4408, Cell Signaling Technology) or polyclonal anti-rabbit Alexa488 (IgG, goat, 1:500) (A-11034, ThermoFisher Scientific) as secondary antibodies. .. In case of staining with SiR-dyes, cells were not permeabilized but directly stained after washing with SiR-Actin (SC001, Spirochrome AG/Tebu-bio) or SiR-DNA (SC007, Spirochrome AG/Tebu-bio) at 3 µM.

    Article Title: PAX6 does not regulate Nfia and Nfib expression during neocortical development
    Article Snippet: For IHC, a biotinylated goat anti-rabbit secondary antibody (1:500; BA-5000, Vector Laboratories) was used, followed by incubation with the VECTASTAIN elite ABC kit (Vector Laboratories) and nickel DAB staining as previously described . .. For IF, Alexa Fluor 488-conjugated goat anti-chicken (1:500; A-11039, Invitrogen) and Alexa Fluor 555-conjugated goat anti-rabbit (1:500; A-11034, Invitrogen) secondary antibodies were used for detection.

    Article Title: Intracellular uptake of macromolecules by brain lymphatic endothelial cells during zebrafish embryonic development
    Article Snippet: Staining was via Cyanine 3 diluted 1:50 in amplification diluent (Perkin Elmer, #SAT704A001EA) for 60 min at room temperature. .. Secondary fluorescent antibodies goat α–Chicken IgG 488 (1:200, Lifetech, #A-11039) and goat α-Rabbit 488 (1:200, Lifetech, #A-11034) were both applied overnight at 4°C.

    Article Title: Phosphatidylserine Exposure Controls Viral Innate Immune Responses by Microglia
    Article Snippet: Cell nuclei were stained using DAPI (1:1000; Thermo Fisher Scientific Cat. # ). .. Secondary antibodies (1:100) included Alexa Fluor 488 goat anti-rabbit (Thermo Fisher Scientific Cat. #A-11034; RRID: AB_2576217), Alexa Fluor 633 goat anti-rat (Thermo Fisher Scientific Cat. #A-21094; RRID: AB_2535749), and Alexa Fluor 633 goat anti-mouse (Thermo Fisher Scientific Cat. #A-21052; RRID: AB_2535719).

    Article Title: Salidroside Inhibits Myogenesis by Modulating p-Smad3-Induced Myf5 Transcription
    Article Snippet: After that, the cells were incubated with a primary antibody against E-MHC (Hybridoma Bank, BF-G6), p-Smad3, Myf5 or myogenin at 4°C overnight (1:200 dilutions), followed by incubation with the Alexa Fluor 594 (Invitrogen, A-11032) fluorescent dye conjugated to an anti-mouse secondary antibody or Alexa Fluor 488 (Invitrogen, A-11034) fluorescent dye conjugated to an anti-rabbit secondary antibody. .. The cells were stained with DAPI to visualize the nuclei.

    other:

    Article Title: Overexpression of MTA1 inhibits the metastatic ability of ZR-75-30 cells in vitro by promoting MTA2 degradation
    Article Snippet: Fluorescent secondary antibodies Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (A-11034) and Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (A-11032) and lipofectamine 3000 (L3000015) were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Article Title: APC2 controls dendrite development by promoting microtubule dynamics
    Article Snippet: Secondary antibodies were used at 1:500 concentrations, unless specified otherwise, as follows: anti-chicken Alexa 488 (Thermo Fisher Scientific, A11039, RRID:AB_142924), anti-mouse Alexa 488 (Thermo Fisher Scientific, A-11029, RRID:AB_2534088; A-21121, RRID:AB_2535764; A-21131 RRID:AB_2535771; A-21141, RRID:AB_2535778), anti-rabbit Alexa 488 (Thermo Fisher Scientific, A-11034, RRID:AB_2576217), anti-rat Alexa 488 (Thermo Fisher Scientific, A-11006, RRID:AB_2534074), anti-mouse Alexa 647 (Thermo Fisher Scientific, A-21240, RRID:AB_2535809; A-21241, RRID:AB_2535810; A-21242, RRID:AB_2535811), anti-rabbit Alexa 647 (Thermo Fisher Scientific, A-21245, RRID:AB_2535813), anti-rat Alexa 647 (Thermo Fisher Scientific, A-21247, RRID:AB_141778), anti-mouse IRdye680LT (1:20,000, LI-COR Biosciences, 926–68020, RRID:AB_10706161), anti-rabbit IRdye680LT (1:20,000, LI-COR Biosciences, 926–68021, RRID:AB_10706309), anti-mouse IRdye800CW (1:15,000, LI-COR Biosciences, 926–32210, RRID:AB_621842), anti-rabbit IRdye800CW (1:15,000, LI-COR Biosciences, 926–32211, RRID:AB_621843).

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    Thermo Scientific MagnaBind Beads provide a convenient method for magnetic separation of antibodies antigens lectins enzymes nucleic acids and cells using affinity binding To remove the MagnaBind Beads from the
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    85
    Thermo Fisher rabbit anti goat igg hrp conjugated antibody
    The immunized mouse serum <t>IgG</t> (H+L) titer and IgG subtypes were detected using the indirect ELISA assay. (A) IgG titers of BALB/c mice immunized with rEF-Tu, rHSP70, Mo extracts and PBS sera of each groups were collected on days 3, 7, 14, 21, 28, 35, 42, 49, 56, 63 and 70 DAI. (B) Determination of IgG subtypes in sera of the immunized mice. The sera of each group were collected at 35 DAI. ELISA plates were coated with purified rEF-Tu proteins or rHSP70 proteins or Mo extracts of M . ovipneumoniae wild strain Mo-1 at a concentration of 100 ng per well. Anti-mouse IgG (H+L) or IgG1 or IgG2a <t>HRP-conjugated</t> antibodies were used as secondary antibodies. Asterisks indicates the results of the One-Way ANOVA using the Tukey test, compared with the PBS negative control group, with P
    Rabbit Anti Goat Igg Hrp Conjugated Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher fitc conjugated goat anti rabbit igg
    Pf MSP8 is expressed during P. falciparum gametocyte development but is absent on the surface of activated macrogametes. (A) Detection of Pf MSP8 expression in fixed and permeabilized P. falciparum gametocytes (stages II to V) by an immunofluorescence assay with rabbit anti-r Pf MSP8 <t>IgG</t> or control IgG followed by <t>FITC-conjugated</t> secondary antibodies. DAPI was used to stain parasite DNA. (B) Analysis of Pf MSP8 expression on activated, live P. falciparum macrogametes by an immunofluorescence assay, as described above, with rabbit anti-r Pf MSP8 IgG. Samples were costained with MAb 4B7, which is specific for Pf s25 (MAb 4B7), followed by TRITC-conjugated secondary IgG. DIC, differential interference contrast.
    Fitc Conjugated Goat Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexa fluor 488 conjugated goat anti rabbit igg secondary antibody
    Determination of AvBD8 protein expression in immune cell lines by performing immunocytochemical analysis. Cultured chicken CU91 T-, DT40 B-, HD11 macrophage-, and OU2 fibroblast-cell lines were incubated with the rabbit anti-AvBD8 primary antibody, followed by incubation with the <t>Alexa</t> Fluor 488-conjugated goat anti-rabbit <t>IgG</t> secondary antibody, and were counterstained with 4′,6-diamidino-2-phenylindole. Control cells were incubated with secondary antibody only. AvBD8, avian beta-defensin 8; IgG, immunoglobulin G. Scale bar: 20 μm.
    Alexa Fluor 488 Conjugated Goat Anti Rabbit Igg Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexa fluor 488 conjugated goat anti rabbit igg secondary antibody/product/Thermo Fisher
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    The immunized mouse serum IgG (H+L) titer and IgG subtypes were detected using the indirect ELISA assay. (A) IgG titers of BALB/c mice immunized with rEF-Tu, rHSP70, Mo extracts and PBS sera of each groups were collected on days 3, 7, 14, 21, 28, 35, 42, 49, 56, 63 and 70 DAI. (B) Determination of IgG subtypes in sera of the immunized mice. The sera of each group were collected at 35 DAI. ELISA plates were coated with purified rEF-Tu proteins or rHSP70 proteins or Mo extracts of M . ovipneumoniae wild strain Mo-1 at a concentration of 100 ng per well. Anti-mouse IgG (H+L) or IgG1 or IgG2a HRP-conjugated antibodies were used as secondary antibodies. Asterisks indicates the results of the One-Way ANOVA using the Tukey test, compared with the PBS negative control group, with P

    Journal: PLoS ONE

    Article Title: Elongation Factor Tu and Heat Shock Protein 70 Are Membrane-Associated Proteins from Mycoplasma ovipneumoniae Capable of Inducing Strong Immune Response in Mice

    doi: 10.1371/journal.pone.0161170

    Figure Lengend Snippet: The immunized mouse serum IgG (H+L) titer and IgG subtypes were detected using the indirect ELISA assay. (A) IgG titers of BALB/c mice immunized with rEF-Tu, rHSP70, Mo extracts and PBS sera of each groups were collected on days 3, 7, 14, 21, 28, 35, 42, 49, 56, 63 and 70 DAI. (B) Determination of IgG subtypes in sera of the immunized mice. The sera of each group were collected at 35 DAI. ELISA plates were coated with purified rEF-Tu proteins or rHSP70 proteins or Mo extracts of M . ovipneumoniae wild strain Mo-1 at a concentration of 100 ng per well. Anti-mouse IgG (H+L) or IgG1 or IgG2a HRP-conjugated antibodies were used as secondary antibodies. Asterisks indicates the results of the One-Way ANOVA using the Tukey test, compared with the PBS negative control group, with P

    Article Snippet: Following washing with PBST three times, membranes were incubated with rabbit anti-goat IgG HRP- conjugated antibody at 37°C for 1 h and exposed to SuperSignal® West Pico Chemiluminescent Substrate reagent for detection (Thermo Scientific, USA).

    Techniques: Indirect ELISA, Mouse Assay, Enzyme-linked Immunosorbent Assay, Purification, Concentration Assay, Negative Control

    Pf MSP8 is expressed during P. falciparum gametocyte development but is absent on the surface of activated macrogametes. (A) Detection of Pf MSP8 expression in fixed and permeabilized P. falciparum gametocytes (stages II to V) by an immunofluorescence assay with rabbit anti-r Pf MSP8 IgG or control IgG followed by FITC-conjugated secondary antibodies. DAPI was used to stain parasite DNA. (B) Analysis of Pf MSP8 expression on activated, live P. falciparum macrogametes by an immunofluorescence assay, as described above, with rabbit anti-r Pf MSP8 IgG. Samples were costained with MAb 4B7, which is specific for Pf s25 (MAb 4B7), followed by TRITC-conjugated secondary IgG. DIC, differential interference contrast.

    Journal: Infection and Immunity

    Article Title: Evaluation of a Plasmodium-Specific Carrier Protein To Enhance Production of Recombinant Pfs25, a Leading Transmission-Blocking Vaccine Candidate

    doi: 10.1128/IAI.00486-17

    Figure Lengend Snippet: Pf MSP8 is expressed during P. falciparum gametocyte development but is absent on the surface of activated macrogametes. (A) Detection of Pf MSP8 expression in fixed and permeabilized P. falciparum gametocytes (stages II to V) by an immunofluorescence assay with rabbit anti-r Pf MSP8 IgG or control IgG followed by FITC-conjugated secondary antibodies. DAPI was used to stain parasite DNA. (B) Analysis of Pf MSP8 expression on activated, live P. falciparum macrogametes by an immunofluorescence assay, as described above, with rabbit anti-r Pf MSP8 IgG. Samples were costained with MAb 4B7, which is specific for Pf s25 (MAb 4B7), followed by TRITC-conjugated secondary IgG. DIC, differential interference contrast.

    Article Snippet: Bound IgG was detected with FITC-conjugated goat anti-rabbit IgG, FITC-conjugated goat anti-mouse IgG, or tetramethylrhodamine isothiocyanate (TRITC)-labeled goat anti-mouse IgG as needed and then analyzed by fluorescence microscopy as described above.

    Techniques: Expressing, Immunofluorescence, Staining

    Determination of AvBD8 protein expression in immune cell lines by performing immunocytochemical analysis. Cultured chicken CU91 T-, DT40 B-, HD11 macrophage-, and OU2 fibroblast-cell lines were incubated with the rabbit anti-AvBD8 primary antibody, followed by incubation with the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody, and were counterstained with 4′,6-diamidino-2-phenylindole. Control cells were incubated with secondary antibody only. AvBD8, avian beta-defensin 8; IgG, immunoglobulin G. Scale bar: 20 μm.

    Journal: Asian-Australasian Journal of Animal Sciences

    Article Title: Expression and regulation of avian beta-defensin 8 protein in immune tissues and cell lines of chickens

    doi: 10.5713/ajas.17.0836

    Figure Lengend Snippet: Determination of AvBD8 protein expression in immune cell lines by performing immunocytochemical analysis. Cultured chicken CU91 T-, DT40 B-, HD11 macrophage-, and OU2 fibroblast-cell lines were incubated with the rabbit anti-AvBD8 primary antibody, followed by incubation with the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody, and were counterstained with 4′,6-diamidino-2-phenylindole. Control cells were incubated with secondary antibody only. AvBD8, avian beta-defensin 8; IgG, immunoglobulin G. Scale bar: 20 μm.

    Article Snippet: After blocking, the cells were incubated overnight with the rabbit anti-AvBD8 primary antibody (dilution, 1:100) at 4°C, followed by incubation with the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (dilution, 1:500) for 1 h. Control cells received secondary antibody only.

    Techniques: Expressing, Cell Culture, Incubation

    Determination of AvBD8 protein expression in immune tissues of male WL chickens by performing immunohistochemical analysis. Immunohistochemical analysis was performed to assess AvBD8 protein expression in the thymus, spleen, liver, small intestine, and ceca of male WL chickens aged 25 weeks. Frozen sections were incubated with the rabbit anti-AvBD8 primary antibody, followed by incubation with the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody, and were counterstained with 4′,6-diamidino-2-phenylindole. Control sections were incubated with secondary antibody only. Boxed region in the middle column is enlarged in the right column. AvBD8, avian beta-defensin 8; WL, White Leghorn; IgG, immunoglobulin G. Scale bar: 200 μm (left and middle columns) and 50 μm (right column). Arrowheads indicate strong AvBD8 signal in the intestinal mucosal layer.

    Journal: Asian-Australasian Journal of Animal Sciences

    Article Title: Expression and regulation of avian beta-defensin 8 protein in immune tissues and cell lines of chickens

    doi: 10.5713/ajas.17.0836

    Figure Lengend Snippet: Determination of AvBD8 protein expression in immune tissues of male WL chickens by performing immunohistochemical analysis. Immunohistochemical analysis was performed to assess AvBD8 protein expression in the thymus, spleen, liver, small intestine, and ceca of male WL chickens aged 25 weeks. Frozen sections were incubated with the rabbit anti-AvBD8 primary antibody, followed by incubation with the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody, and were counterstained with 4′,6-diamidino-2-phenylindole. Control sections were incubated with secondary antibody only. Boxed region in the middle column is enlarged in the right column. AvBD8, avian beta-defensin 8; WL, White Leghorn; IgG, immunoglobulin G. Scale bar: 200 μm (left and middle columns) and 50 μm (right column). Arrowheads indicate strong AvBD8 signal in the intestinal mucosal layer.

    Article Snippet: After blocking, the cells were incubated overnight with the rabbit anti-AvBD8 primary antibody (dilution, 1:100) at 4°C, followed by incubation with the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (dilution, 1:500) for 1 h. Control cells received secondary antibody only.

    Techniques: Expressing, Immunohistochemistry, Incubation