alexa fluor 488 conjugated goat anti mouse igg  (Thermo Fisher)


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    Name:
    Alexa Fluor 488 Goat Anti Mouse SFX Kit
    Description:
    Alexa Fluor SFX Kits contain Image iT FX signal enhancer Cat no I36933 plus one of sixteen different Alexa Fluor dye labeled secondary antibodies These kits provide 400 µg 0 2 mL of 2 mg mL of either goat anti mouse IgG or goat anti rabbit IgG antibody as a standard or highly cross adsorbed preparation conjugated to Alexa Fluor 488 Alexa Fluor 555 Alexa Fluor 594 or Alexa Fluor 647 dye four of our most commonly used Alexa Fluor dyes The Alexa Fluor 488 goat anti mouse IgG highly cross adsorbed included with this kit is also available in a 1000 µg unit size Cat no A11029
    Catalog Number:
    A31620
    Price:
    None
    Category:
    Antibodies Secondary Detection Reagents
    Applications:
    Antibodies and Secondary Detection|Cell Analysis
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    Structured Review

    Thermo Fisher alexa fluor 488 conjugated goat anti mouse igg
    Treatment with iloprost prevents LPC-mediated vascular barrier disruption in the adult spinal cord. A , visualization of vascular leakage in the CNS. Upper panels, representative images of the thoracic spinal cord showing leakage of <t>Alexa</t> Fluor 555-conjugated cadaverine ( red ) into the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. The broken line represents the outline of the tissue. Scale bar, 1 mm. Middle panels, representative images of the thoracic spinal cord showing the distribution of Alexa Fluor 555-conjugated cadaverine ( red ) in the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. Sections were counterstained for CD31 (vascular endothelial cell marker, green ). Scale bar, 100 μm. Lower panels, representative images of cross-sections of the thoracic spinal cord showing leakage of endogenous <t>IgG</t> ( green ) into the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. Sections were vascular counterstained with DyLight 594-labeled L. esculentum lectin (vascular endothelial cell marker, red ). Scale bar, 100 μm. B , quantification of Evans Blue leakage in lesions of the spinal cord at 1 day after the operation. Values represent the mean ± S.E. of three independent experiments. **, p
    Alexa Fluor SFX Kits contain Image iT FX signal enhancer Cat no I36933 plus one of sixteen different Alexa Fluor dye labeled secondary antibodies These kits provide 400 µg 0 2 mL of 2 mg mL of either goat anti mouse IgG or goat anti rabbit IgG antibody as a standard or highly cross adsorbed preparation conjugated to Alexa Fluor 488 Alexa Fluor 555 Alexa Fluor 594 or Alexa Fluor 647 dye four of our most commonly used Alexa Fluor dyes The Alexa Fluor 488 goat anti mouse IgG highly cross adsorbed included with this kit is also available in a 1000 µg unit size Cat no A11029
    https://www.bioz.com/result/alexa fluor 488 conjugated goat anti mouse igg/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    alexa fluor 488 conjugated goat anti mouse igg - by Bioz Stars, 2021-06
    99/100 stars

    Images

    1) Product Images from "Prostacyclin Prevents Pericyte Loss and Demyelination Induced by Lysophosphatidylcholine in the Central Nervous System *"

    Article Title: Prostacyclin Prevents Pericyte Loss and Demyelination Induced by Lysophosphatidylcholine in the Central Nervous System *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.587253

    Treatment with iloprost prevents LPC-mediated vascular barrier disruption in the adult spinal cord. A , visualization of vascular leakage in the CNS. Upper panels, representative images of the thoracic spinal cord showing leakage of Alexa Fluor 555-conjugated cadaverine ( red ) into the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. The broken line represents the outline of the tissue. Scale bar, 1 mm. Middle panels, representative images of the thoracic spinal cord showing the distribution of Alexa Fluor 555-conjugated cadaverine ( red ) in the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. Sections were counterstained for CD31 (vascular endothelial cell marker, green ). Scale bar, 100 μm. Lower panels, representative images of cross-sections of the thoracic spinal cord showing leakage of endogenous IgG ( green ) into the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. Sections were vascular counterstained with DyLight 594-labeled L. esculentum lectin (vascular endothelial cell marker, red ). Scale bar, 100 μm. B , quantification of Evans Blue leakage in lesions of the spinal cord at 1 day after the operation. Values represent the mean ± S.E. of three independent experiments. **, p
    Figure Legend Snippet: Treatment with iloprost prevents LPC-mediated vascular barrier disruption in the adult spinal cord. A , visualization of vascular leakage in the CNS. Upper panels, representative images of the thoracic spinal cord showing leakage of Alexa Fluor 555-conjugated cadaverine ( red ) into the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. The broken line represents the outline of the tissue. Scale bar, 1 mm. Middle panels, representative images of the thoracic spinal cord showing the distribution of Alexa Fluor 555-conjugated cadaverine ( red ) in the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. Sections were counterstained for CD31 (vascular endothelial cell marker, green ). Scale bar, 100 μm. Lower panels, representative images of cross-sections of the thoracic spinal cord showing leakage of endogenous IgG ( green ) into the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. Sections were vascular counterstained with DyLight 594-labeled L. esculentum lectin (vascular endothelial cell marker, red ). Scale bar, 100 μm. B , quantification of Evans Blue leakage in lesions of the spinal cord at 1 day after the operation. Values represent the mean ± S.E. of three independent experiments. **, p

    Techniques Used: Injection, Marker, Labeling

    2) Product Images from "Salinomycin Inhibits Influenza Virus Infection by Disrupting Endosomal Acidification and Viral Matrix Protein 2 Function"

    Article Title: Salinomycin Inhibits Influenza Virus Infection by Disrupting Endosomal Acidification and Viral Matrix Protein 2 Function

    Journal: Journal of Virology

    doi: 10.1128/JVI.01441-18

    Effects of salinomycin on the early stages of the influenza virus life cycle. (A) Time-of-addition experiments. The experimental process is described on the left. MDCK cells were infected with influenza PR8 virus for 1 h at 4°C. After removal of unadsorbed virus, the cells were incubated for an additional 4 h at 35°C. They were inoculated under different conditions, i.e., in the absence or presence of 10 µM salinomycin or EGCG. In parallel, at 1, 2, and 4 h p.i., the compounds were added to the cell culture medium. At 5 h p.i., the cell monolayers were washed with PBS and overlay medium was added to allow plaque generation. The numbers are expressed as percentages relative to plaque number from the DMSO-treated sample and represent the means ± SD of triplicate samples. (B) Confocal microscopy showing the subcellular distribution of viral NP. MDCK cells were mock infected (no virus) or infected with PR8 virus at an MOI of 2.5 for 4 h at 37°C in the presence of DMSO, salinomycin, EGCG, or RBV. The viral NP protein was detected using an NP-specific monoclonal antibody and an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (green). Nuclei were counterstained with DAPI (blue). Original magnification, ×400.
    Figure Legend Snippet: Effects of salinomycin on the early stages of the influenza virus life cycle. (A) Time-of-addition experiments. The experimental process is described on the left. MDCK cells were infected with influenza PR8 virus for 1 h at 4°C. After removal of unadsorbed virus, the cells were incubated for an additional 4 h at 35°C. They were inoculated under different conditions, i.e., in the absence or presence of 10 µM salinomycin or EGCG. In parallel, at 1, 2, and 4 h p.i., the compounds were added to the cell culture medium. At 5 h p.i., the cell monolayers were washed with PBS and overlay medium was added to allow plaque generation. The numbers are expressed as percentages relative to plaque number from the DMSO-treated sample and represent the means ± SD of triplicate samples. (B) Confocal microscopy showing the subcellular distribution of viral NP. MDCK cells were mock infected (no virus) or infected with PR8 virus at an MOI of 2.5 for 4 h at 37°C in the presence of DMSO, salinomycin, EGCG, or RBV. The viral NP protein was detected using an NP-specific monoclonal antibody and an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (green). Nuclei were counterstained with DAPI (blue). Original magnification, ×400.

    Techniques Used: Infection, Incubation, Cell Culture, Confocal Microscopy

    Endosomal escape of vRNP is affected by salinomycin. Influenza virus PR8-infected A549 cells (MOI, 10) were treated for 8 h with DMSO (mock) or 10 μM salinomycin in MEM supplemented with 10 μg/ml of cycloheximide. Cells were then costained for viral NP and the early endosomal marker EEA1 (A) or the late endosomal marker LAMP1 (B). NP protein was visualized with an anti-NP antibody, followed by an Alexa Fluor 633-conjugated secondary antibody (red). EEA1 and LAMP1 were detected using their specific antibodies, followed by an Alexa Fluor 488-conjugated secondary antibody (green). Nuclei were counterstained with DAPI (blue). Original magnification, ×630.
    Figure Legend Snippet: Endosomal escape of vRNP is affected by salinomycin. Influenza virus PR8-infected A549 cells (MOI, 10) were treated for 8 h with DMSO (mock) or 10 μM salinomycin in MEM supplemented with 10 μg/ml of cycloheximide. Cells were then costained for viral NP and the early endosomal marker EEA1 (A) or the late endosomal marker LAMP1 (B). NP protein was visualized with an anti-NP antibody, followed by an Alexa Fluor 633-conjugated secondary antibody (red). EEA1 and LAMP1 were detected using their specific antibodies, followed by an Alexa Fluor 488-conjugated secondary antibody (green). Nuclei were counterstained with DAPI (blue). Original magnification, ×630.

    Techniques Used: Infection, Marker

    3) Product Images from "Human Cytomegalovirus Immediate-Early 1 Protein Rewires Upstream STAT3 to Downstream STAT1 Signaling Switching an IL6-Type to an IFNγ-Like Response"

    Article Title: Human Cytomegalovirus Immediate-Early 1 Protein Rewires Upstream STAT3 to Downstream STAT1 Signaling Switching an IL6-Type to an IFNγ-Like Response

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005748

    Residues within IE1 region 410–445 are required for targeting of STAT3 and down-regulation of STAT3-responsive genes. (A) TetR cells without (w/o) or with inducible expression of the indicated HA-IE1 proteins were treated with dox for 48 h. During the final 24 h of dox treatment, cells were kept in medium with 0.5% FBS. Subcellular localization of endogenous STAT3α in IE1 expressing cells was analyzed by indirect immunofluorescence microscopy. Samples were simultaneously reacted with a rabbit monoclonal antibody to STAT3α and a mouse monoclonal antibody to HA-tagged IE1, followed by incubation with a rabbit-specific Alexa Fluor 594 conjugate and a mouse-specific Alexa Fluor 488 conjugate. Host cell nuclei were visualized by 4',6-diamidino-2-phenylindole (DAPI) staining. Additionally, merge images of STAT3α, IE1 and DAPI signals are presented. (B) The percentage of cells with i) predominantly nuclear STAT3α staining (N > C), ii) equally strong nuclear and cytoplasmic STAT3α staining (N = C) and iii) predominantly cytoplasmic STAT3α staining (C > N) was determined for 100 randomly selected cells per sample described in (A). (C) TetR cells without or with inducible expression of HA-tagged wild-type IE1 or IE1dl410-420 were treated with dox for 72 h and with solvent (w/o) or IL6 plus IL6R (IL6/Rα) for 30 min. Cytoplasmic and nuclear extracts were prepared and analyzed by immunoblotting for histone H2B, STAT2, STAT3α and IE1. (D) TetR cells without (w/o) or with inducible expression of HA-tagged wild-type IE1 or IE1dl410-420 were treated with dox for 72 h. Whole cell extracts were prepared and used for immunoprecipitations (IPs) with anti-HA-agarose. Samples of lysates and immunoprecipitates were analyzed by immunoblotting for IE1 and STAT3α. (E) TetR cells without (w/o) or with inducible expression of HA-tagged wild-type IE1 or IE1dl410-420 were treated with dox for 72 h and with IL6 plus IL6R for 30 min. Samples were subjected to ChIP with rabbit polyclonal antibodies to STAT3 or normal rabbit IgG and primers specific for sequences in the SOCS3 promoter or coding region. The percentage of output versus input DNA is presented as the difference between STAT3 and normal IgG ChIPs. Means and standard deviations of two biological and two technical replicates are shown. (F) TetR cells without (w/o) or with inducible expression of the indicated HA-tagged wild-type or mutant IE1 proteins were treated with dox for 72 h. Relative mRNA expression levels were determined by RT-qPCR with primers specific for the STAT3 target genes CXCL12 and SOCS3. Results were normalized to TUBB, and means and standard deviations of two biological and two technical replicates are shown in comparison to IE1-negative TetR cells (set to 1).
    Figure Legend Snippet: Residues within IE1 region 410–445 are required for targeting of STAT3 and down-regulation of STAT3-responsive genes. (A) TetR cells without (w/o) or with inducible expression of the indicated HA-IE1 proteins were treated with dox for 48 h. During the final 24 h of dox treatment, cells were kept in medium with 0.5% FBS. Subcellular localization of endogenous STAT3α in IE1 expressing cells was analyzed by indirect immunofluorescence microscopy. Samples were simultaneously reacted with a rabbit monoclonal antibody to STAT3α and a mouse monoclonal antibody to HA-tagged IE1, followed by incubation with a rabbit-specific Alexa Fluor 594 conjugate and a mouse-specific Alexa Fluor 488 conjugate. Host cell nuclei were visualized by 4',6-diamidino-2-phenylindole (DAPI) staining. Additionally, merge images of STAT3α, IE1 and DAPI signals are presented. (B) The percentage of cells with i) predominantly nuclear STAT3α staining (N > C), ii) equally strong nuclear and cytoplasmic STAT3α staining (N = C) and iii) predominantly cytoplasmic STAT3α staining (C > N) was determined for 100 randomly selected cells per sample described in (A). (C) TetR cells without or with inducible expression of HA-tagged wild-type IE1 or IE1dl410-420 were treated with dox for 72 h and with solvent (w/o) or IL6 plus IL6R (IL6/Rα) for 30 min. Cytoplasmic and nuclear extracts were prepared and analyzed by immunoblotting for histone H2B, STAT2, STAT3α and IE1. (D) TetR cells without (w/o) or with inducible expression of HA-tagged wild-type IE1 or IE1dl410-420 were treated with dox for 72 h. Whole cell extracts were prepared and used for immunoprecipitations (IPs) with anti-HA-agarose. Samples of lysates and immunoprecipitates were analyzed by immunoblotting for IE1 and STAT3α. (E) TetR cells without (w/o) or with inducible expression of HA-tagged wild-type IE1 or IE1dl410-420 were treated with dox for 72 h and with IL6 plus IL6R for 30 min. Samples were subjected to ChIP with rabbit polyclonal antibodies to STAT3 or normal rabbit IgG and primers specific for sequences in the SOCS3 promoter or coding region. The percentage of output versus input DNA is presented as the difference between STAT3 and normal IgG ChIPs. Means and standard deviations of two biological and two technical replicates are shown. (F) TetR cells without (w/o) or with inducible expression of the indicated HA-tagged wild-type or mutant IE1 proteins were treated with dox for 72 h. Relative mRNA expression levels were determined by RT-qPCR with primers specific for the STAT3 target genes CXCL12 and SOCS3. Results were normalized to TUBB, and means and standard deviations of two biological and two technical replicates are shown in comparison to IE1-negative TetR cells (set to 1).

    Techniques Used: Expressing, Immunofluorescence, Microscopy, Incubation, Staining, Chromatin Immunoprecipitation, Mutagenesis, Quantitative RT-PCR

    4) Product Images from "Polarity Changes in the Transmembrane Domain Core of HIV-1 Vpu Inhibits Its Anti-Tetherin Activity"

    Article Title: Polarity Changes in the Transmembrane Domain Core of HIV-1 Vpu Inhibits Its Anti-Tetherin Activity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0020890

    Effects of Vpu TM mutations on Vpu-mediated degradation and surface downregulation of tetherin. (A) 293T cells were co-transfected with 100 ng HA-tetherin expression plasmid along with 200 ng VR1012 control vector or VR1012 encoding Vpu TM variants at a 2∶1 molar ratio. At 48 h post-transfection, the cells were harvested for immunoblotting analysis. Tetherin and Vpu were detected with anti-HA and anti-myc antibody, respectively. Tubulin was detected as a loading control. (B) Tetherin levels were measured using Bandscan software and normalized by tubulin levels. Percentages of degraded tetherin were calculated by subtracting the densitometric intensity values of the indicated Vpu WT or mutant bands from that of the mock band to represent the different abilities of Vpu variants to mediate tetherin degradation. Values are representative of three independent experiments. (C) HeLa cells were co-transfected with 500 ng pEGFP-N3 along with 500 ng VR1012 control vector or VR1012 encoding Vpu TM variants. Cell surface tetherin was stained with BST-2 antibodies, followed by Alexa 633 goat anti-mouse IgG and analyzed by flow cytometry. Samples were gated on EGFP+ cells, and the surface tetherin levels are shown in the histograms with median values at the top right corner.
    Figure Legend Snippet: Effects of Vpu TM mutations on Vpu-mediated degradation and surface downregulation of tetherin. (A) 293T cells were co-transfected with 100 ng HA-tetherin expression plasmid along with 200 ng VR1012 control vector or VR1012 encoding Vpu TM variants at a 2∶1 molar ratio. At 48 h post-transfection, the cells were harvested for immunoblotting analysis. Tetherin and Vpu were detected with anti-HA and anti-myc antibody, respectively. Tubulin was detected as a loading control. (B) Tetherin levels were measured using Bandscan software and normalized by tubulin levels. Percentages of degraded tetherin were calculated by subtracting the densitometric intensity values of the indicated Vpu WT or mutant bands from that of the mock band to represent the different abilities of Vpu variants to mediate tetherin degradation. Values are representative of three independent experiments. (C) HeLa cells were co-transfected with 500 ng pEGFP-N3 along with 500 ng VR1012 control vector or VR1012 encoding Vpu TM variants. Cell surface tetherin was stained with BST-2 antibodies, followed by Alexa 633 goat anti-mouse IgG and analyzed by flow cytometry. Samples were gated on EGFP+ cells, and the surface tetherin levels are shown in the histograms with median values at the top right corner.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Software, Mutagenesis, Staining, Flow Cytometry, Cytometry

    Effects of Vpu TM mutations on Vpu-mediated degradation and surface downregulation of CD4. (A) 293T cells were co-transfected with 100 ng CD4-HA expression plasmid along with 200 ng VR1012 control vector or VR1012 encoding Vpu TM variants. At 48 h post-transfection, the cells were harvested for immunoblotting analysis. CD4 and Vpu were detected with anti-HA and anti-myc antibodies, respectively. Tubulin was detected as a loading control. (B) CD4 levels were measured using Bandscan software and normalized by tubulin levels. Percentages of degraded CD4 were calculated by subtracting the densitometric intensity values of the indicated Vpu WT or mutant bands from that of the mock band to represent the different abilities of Vpu variants to mediate CD4 degradation. Results shown are the average of two independent experiments. (C) HeLa CD4 cells were co-transfected with 500 ng pEGFP-N3 along with 500 ng VR1012 control vector or VR1012 encoding Vpu TM variants. Cell surface CD4 was stained with CD4 antibodies followed by Alexa 633 goat anti-mouse IgG and analyzed by flow cytometry. Samples were gated on EGFP+ cells, and the surface CD4 levels are shown in the histograms with median values at the top right corner.
    Figure Legend Snippet: Effects of Vpu TM mutations on Vpu-mediated degradation and surface downregulation of CD4. (A) 293T cells were co-transfected with 100 ng CD4-HA expression plasmid along with 200 ng VR1012 control vector or VR1012 encoding Vpu TM variants. At 48 h post-transfection, the cells were harvested for immunoblotting analysis. CD4 and Vpu were detected with anti-HA and anti-myc antibodies, respectively. Tubulin was detected as a loading control. (B) CD4 levels were measured using Bandscan software and normalized by tubulin levels. Percentages of degraded CD4 were calculated by subtracting the densitometric intensity values of the indicated Vpu WT or mutant bands from that of the mock band to represent the different abilities of Vpu variants to mediate CD4 degradation. Results shown are the average of two independent experiments. (C) HeLa CD4 cells were co-transfected with 500 ng pEGFP-N3 along with 500 ng VR1012 control vector or VR1012 encoding Vpu TM variants. Cell surface CD4 was stained with CD4 antibodies followed by Alexa 633 goat anti-mouse IgG and analyzed by flow cytometry. Samples were gated on EGFP+ cells, and the surface CD4 levels are shown in the histograms with median values at the top right corner.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Software, Mutagenesis, Staining, Flow Cytometry, Cytometry

    5) Product Images from "Antifungal Activity of Mammalian Serum Amyloid A1 against Candida albicans"

    Article Title: Antifungal Activity of Mammalian Serum Amyloid A1 against Candida albicans

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.01975-19

    rhSAA1 binds to the cell surface of C. albicans and causes cell membrane damage. (a) Immunofluorescence staining assays. Yeast-form and filamentous cells of C. albicans (SC5314) were treated with or without 200 mg/liter rhSAA1. Anti-human SAA1 antibody and Alexa Fluor 488-conjugated anti-mouse IgG were used for immunofluorescence staining assays. (b) Fluorescence-activated cell sorting analysis of PI uptake. Yeast-form cells of C. albicans (SC5314, 5 × 10 6 cells/ml) were treated with 200 mg/liter rhSAA1 for 30 min or 60 min and stained with the red dye PI. The cells were then subjected to flow cytometry analysis. The PBS and amphotericin B (AMP B) treatments served as negative and positive controls, respectively. SSC-A, side scatter area; PI-A, propidium iodide area. The percentages of cells stained with PI are expressed as means ± the SD of three independent experiments. (c) Cellular images of PI staining assays. C. albicans yeast-form cells (5 × 10 6 cells/ml) were treated with 200 mg/liter rhSAA1 for 60 min and stained with PI. The PBS and amphotericin B (AMP B) treatments served as negative and positive controls, respectively. The percentages of stained cells are shown in the corresponding images. (d) TEM assays. C. albicans cells treated with or without 200 mg/liter rhSAA1 for 3 h were used. The red arrow indicates membrane damage.
    Figure Legend Snippet: rhSAA1 binds to the cell surface of C. albicans and causes cell membrane damage. (a) Immunofluorescence staining assays. Yeast-form and filamentous cells of C. albicans (SC5314) were treated with or without 200 mg/liter rhSAA1. Anti-human SAA1 antibody and Alexa Fluor 488-conjugated anti-mouse IgG were used for immunofluorescence staining assays. (b) Fluorescence-activated cell sorting analysis of PI uptake. Yeast-form cells of C. albicans (SC5314, 5 × 10 6 cells/ml) were treated with 200 mg/liter rhSAA1 for 30 min or 60 min and stained with the red dye PI. The cells were then subjected to flow cytometry analysis. The PBS and amphotericin B (AMP B) treatments served as negative and positive controls, respectively. SSC-A, side scatter area; PI-A, propidium iodide area. The percentages of cells stained with PI are expressed as means ± the SD of three independent experiments. (c) Cellular images of PI staining assays. C. albicans yeast-form cells (5 × 10 6 cells/ml) were treated with 200 mg/liter rhSAA1 for 60 min and stained with PI. The PBS and amphotericin B (AMP B) treatments served as negative and positive controls, respectively. The percentages of stained cells are shown in the corresponding images. (d) TEM assays. C. albicans cells treated with or without 200 mg/liter rhSAA1 for 3 h were used. The red arrow indicates membrane damage.

    Techniques Used: Immunofluorescence, Staining, Fluorescence, FACS, Flow Cytometry, Transmission Electron Microscopy

    6) Product Images from "The DUBm subunit Sgf11 is required for mRNA export and interacts with Cbp80 in Drosophila"

    Article Title: The DUBm subunit Sgf11 is required for mRNA export and interacts with Cbp80 in Drosophila

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks857

    Sgf11 is associated with mRNAs of several genes, and its RNAi knockdown interferes with general mRNA export. ( A ) RIP experiments with hsp70 mRNA after heat shock were performed using antibodies against Sgf11 or components of the mRNA-interacting AMEX complex (ENY2, Xmas-2); nonimmune IgG was used as control. The results are shown as a percentage of input. ( B ) Sgf11 binds to mRNAs of Ras and tubulin genes under normal conditions. The U1 snRNA was used as a control. Antibodies used in RIP experiments were the same as in Figure 3 A. The results are shown as a percentage of input. ( C ) The level of Sgf11, Nonstop and Xmas-2 knockdown in experiments shown in Figures 3 D–3G as estimated by western blot analysis in cells treated with GFP dsRNA (control) or dsRNA corresponding to Sgf11 and Nonstop. Tubulin was used as a loading control. ( D ) RNAi knockdown of Sgf11, but not Nonstop, interferes with hsp70 mRNA export after heat shock. Cells were treated with GFP dsRNA (control) or dsRNA corresponding to Sgf11 and Nonstop. Xmas-2 RNAi knockdown was performed as a positive control. Representative examples of the distribution of hsp70 mRNA (green staining) and cell nuclei (red staining) and corresponding merged images are shown for control cells and cells after Sgf11, Nonstop or Xmas-2 knockdown (magnification, ×1000). The hsp70 transcript was detected by RNA FISH using an Alexa 488-labeled probe; the nuclei were stained with DAPI. The images were recolored in Photoshop for better visualization. ( E ) Quantitative presentation of the results of experiments shown on Figure 3 D. Bars show the percentage of cells with disturbed hsp70 mRNA nuclear export (about 200 cells per RNAi experiment were examined). ( F ) RNAi knockdown of Sgf11, but not Nonstop, interferes with general mRNA export. Cells were treated with GFP dsRNA (control) or dsRNA corresponding to Sgf11 and Nonstop. Xmas-2 RNAi knockdown was performed as a positive control. Representative examples of the distribution of mRNA (green staining) and cell nuclei (red staining) and corresponding merged images are shown for control cells and cells after Sgf11 or Nonstop knockdown (magnification, ×1000). RNA FISH was carried out using a Cy3-labeled oligo(dT) probe to identify poly(A) RNA. The nuclei were stained blue with DAPI. The images were recolored in Photoshop for better visualization. ( G ) Quantitative presentation of the results of experiments shown in Figure 3 F. Bars show the percentage of cells with disturbed hsp70 mRNA nuclear export (about 200 cells per RNAi experiment were examined).
    Figure Legend Snippet: Sgf11 is associated with mRNAs of several genes, and its RNAi knockdown interferes with general mRNA export. ( A ) RIP experiments with hsp70 mRNA after heat shock were performed using antibodies against Sgf11 or components of the mRNA-interacting AMEX complex (ENY2, Xmas-2); nonimmune IgG was used as control. The results are shown as a percentage of input. ( B ) Sgf11 binds to mRNAs of Ras and tubulin genes under normal conditions. The U1 snRNA was used as a control. Antibodies used in RIP experiments were the same as in Figure 3 A. The results are shown as a percentage of input. ( C ) The level of Sgf11, Nonstop and Xmas-2 knockdown in experiments shown in Figures 3 D–3G as estimated by western blot analysis in cells treated with GFP dsRNA (control) or dsRNA corresponding to Sgf11 and Nonstop. Tubulin was used as a loading control. ( D ) RNAi knockdown of Sgf11, but not Nonstop, interferes with hsp70 mRNA export after heat shock. Cells were treated with GFP dsRNA (control) or dsRNA corresponding to Sgf11 and Nonstop. Xmas-2 RNAi knockdown was performed as a positive control. Representative examples of the distribution of hsp70 mRNA (green staining) and cell nuclei (red staining) and corresponding merged images are shown for control cells and cells after Sgf11, Nonstop or Xmas-2 knockdown (magnification, ×1000). The hsp70 transcript was detected by RNA FISH using an Alexa 488-labeled probe; the nuclei were stained with DAPI. The images were recolored in Photoshop for better visualization. ( E ) Quantitative presentation of the results of experiments shown on Figure 3 D. Bars show the percentage of cells with disturbed hsp70 mRNA nuclear export (about 200 cells per RNAi experiment were examined). ( F ) RNAi knockdown of Sgf11, but not Nonstop, interferes with general mRNA export. Cells were treated with GFP dsRNA (control) or dsRNA corresponding to Sgf11 and Nonstop. Xmas-2 RNAi knockdown was performed as a positive control. Representative examples of the distribution of mRNA (green staining) and cell nuclei (red staining) and corresponding merged images are shown for control cells and cells after Sgf11 or Nonstop knockdown (magnification, ×1000). RNA FISH was carried out using a Cy3-labeled oligo(dT) probe to identify poly(A) RNA. The nuclei were stained blue with DAPI. The images were recolored in Photoshop for better visualization. ( G ) Quantitative presentation of the results of experiments shown in Figure 3 F. Bars show the percentage of cells with disturbed hsp70 mRNA nuclear export (about 200 cells per RNAi experiment were examined).

    Techniques Used: Western Blot, Positive Control, Staining, Fluorescence In Situ Hybridization, Labeling

    7) Product Images from "Outer Membrane Vesicles from the Probiotic Escherichia coli Nissle 1917 and the Commensal ECOR12 Enter Intestinal Epithelial Cells via Clathrin-Dependent Endocytosis and Elicit Differential Effects on DNA Damage"

    Article Title: Outer Membrane Vesicles from the Probiotic Escherichia coli Nissle 1917 and the Commensal ECOR12 Enter Intestinal Epithelial Cells via Clathrin-Dependent Endocytosis and Elicit Differential Effects on DNA Damage

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0160374

    Colocalization of EcN and ECOR12 OMVs with clathrin (A), endosomes (B) and lysosomes (C). HT-29 cells were incubated with rhodamine B-R18-labeled OMVs (2 μg) for the indicated times and analyzed using laser scanning confocal spectral microscope. Scale bar: 20 μm. Clathrin was stained using anti-clathrin mouse monoclonal antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG (green). Endosomes were labeled with a rabbit polyclonal antibody against the endosome-associated protein EEA1 and Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). Lysosomes were detected using LysoTracker Green DND-26 at 300 nM (green). Images are from a single representative experiment (n = 4). Colocalization of the green (clathrin, EEA1 or the LysoTracker probe)) and red (vesicles) signals was confirmed by histogram analysis of the fluorescence intensities along the yellow lines. Analysis by laser scanning confocal spectral microscope was performed as described for Fig 1 .
    Figure Legend Snippet: Colocalization of EcN and ECOR12 OMVs with clathrin (A), endosomes (B) and lysosomes (C). HT-29 cells were incubated with rhodamine B-R18-labeled OMVs (2 μg) for the indicated times and analyzed using laser scanning confocal spectral microscope. Scale bar: 20 μm. Clathrin was stained using anti-clathrin mouse monoclonal antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG (green). Endosomes were labeled with a rabbit polyclonal antibody against the endosome-associated protein EEA1 and Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). Lysosomes were detected using LysoTracker Green DND-26 at 300 nM (green). Images are from a single representative experiment (n = 4). Colocalization of the green (clathrin, EEA1 or the LysoTracker probe)) and red (vesicles) signals was confirmed by histogram analysis of the fluorescence intensities along the yellow lines. Analysis by laser scanning confocal spectral microscope was performed as described for Fig 1 .

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

    8) Product Images from "The binding property of a monoclonal antibody against the extracellular domains of aquaporin-4 directs aquaporin-4 toward endocytosis"

    Article Title: The binding property of a monoclonal antibody against the extracellular domains of aquaporin-4 directs aquaporin-4 toward endocytosis

    Journal: Biochemistry and Biophysics Reports

    doi: 10.1016/j.bbrep.2016.05.017

    Cluster formation of AQP4 by binding of E5415A on an astrocytic membrane. Primary cultured astrocytes treated with E5415A (A, C, E, and G) or E5415B (B, D, F, and H) for 10 min (A and B), 1 h (C and D), or 3 h (E and F), or 24 h (G and H) at 37 °C were cooled to 4 °C and further treated with Alexa-Fluor-488-labeled anti-mouse IgG (green) at 4 °C for 1 h. Then cells were fixed with 4% PFA and permeabilized to detect intracellular mAb with Alexa-Fluor-555-labeled anti-mouse IgG (magenta). Bar=5 µm. The lower panels are global images of the upper panels. Magnified areas are indicated by white boxes. Bar=10 µm. (I) Quantification of the number of dots on the cell surface of primary cultured astrocytes incubated with E5415A (red bars) or E5415B (blue bars) followed by visualized with Alexa-Fluor-488-labeled anti-mouse IgG. Fifteen to twenty one images from five cells were taken in each experiment. Numbers of dots in 400 µm 2 were counted using ImageJ2 (National Institute of Health, Bertesda, MD, http://imagej.nih.gov/ij/ ) software. Values are means±SD of four independent experiments. **( P
    Figure Legend Snippet: Cluster formation of AQP4 by binding of E5415A on an astrocytic membrane. Primary cultured astrocytes treated with E5415A (A, C, E, and G) or E5415B (B, D, F, and H) for 10 min (A and B), 1 h (C and D), or 3 h (E and F), or 24 h (G and H) at 37 °C were cooled to 4 °C and further treated with Alexa-Fluor-488-labeled anti-mouse IgG (green) at 4 °C for 1 h. Then cells were fixed with 4% PFA and permeabilized to detect intracellular mAb with Alexa-Fluor-555-labeled anti-mouse IgG (magenta). Bar=5 µm. The lower panels are global images of the upper panels. Magnified areas are indicated by white boxes. Bar=10 µm. (I) Quantification of the number of dots on the cell surface of primary cultured astrocytes incubated with E5415A (red bars) or E5415B (blue bars) followed by visualized with Alexa-Fluor-488-labeled anti-mouse IgG. Fifteen to twenty one images from five cells were taken in each experiment. Numbers of dots in 400 µm 2 were counted using ImageJ2 (National Institute of Health, Bertesda, MD, http://imagej.nih.gov/ij/ ) software. Values are means±SD of four independent experiments. **( P

    Techniques Used: Binding Assay, Cell Culture, Labeling, Incubation, Software

    Induction of endocytosis of AQP4 by mAbs in primary cultured astrocytes. (A and B) Localization of fluorescence-labeled E5415A (A, magenta) or E5415B (B, magenta) and Alexa-Fluor-488-labeled transferrin (green) in primary cultured astrocytes was examined by confocal microscopy up to 3 h. Bar=5 µm. (C and D) Localization of fluorescence-labeled E5415A (C, magenta) or E5415B (D, magenta) and Lysotracker 488 (green) in primary cultured astrocytes was examined by confocal microscopy after incubation for 12 and 24 h. Bar=10 µm.
    Figure Legend Snippet: Induction of endocytosis of AQP4 by mAbs in primary cultured astrocytes. (A and B) Localization of fluorescence-labeled E5415A (A, magenta) or E5415B (B, magenta) and Alexa-Fluor-488-labeled transferrin (green) in primary cultured astrocytes was examined by confocal microscopy up to 3 h. Bar=5 µm. (C and D) Localization of fluorescence-labeled E5415A (C, magenta) or E5415B (D, magenta) and Lysotracker 488 (green) in primary cultured astrocytes was examined by confocal microscopy after incubation for 12 and 24 h. Bar=10 µm.

    Techniques Used: Cell Culture, Fluorescence, Labeling, Confocal Microscopy, Incubation

    9) Product Images from "The Guanine Nucleotide Exchange Protein for ADP-ribosylation Factor 6, ARF-GEP100/BRAG2, Regulates Phagocytosis of Monocytic Phagocytes in an ARF6-dependent Process *"

    Article Title: The Guanine Nucleotide Exchange Protein for ADP-ribosylation Factor 6, ARF-GEP100/BRAG2, Regulates Phagocytosis of Monocytic Phagocytes in an ARF6-dependent Process *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.107458

    Distribution of GEP100 during phagocytosis. PMA-differentiated U937 cells were incubated without ( Resting ) or with opsonized zymosan or IgG beads as in . After fixation, cells were reacted with rabbit anti-GEP100 antibody followed by Alexa 594-labeled
    Figure Legend Snippet: Distribution of GEP100 during phagocytosis. PMA-differentiated U937 cells were incubated without ( Resting ) or with opsonized zymosan or IgG beads as in . After fixation, cells were reacted with rabbit anti-GEP100 antibody followed by Alexa 594-labeled

    Techniques Used: Incubation, Labeling

    10) Product Images from "Characterization and expression analysis of a newly identified glutathione S-transferase of the hard tick Haemaphysalis longicornis during blood-feeding"

    Article Title: Characterization and expression analysis of a newly identified glutathione S-transferase of the hard tick Haemaphysalis longicornis during blood-feeding

    Journal: Parasites & Vectors

    doi: 10.1186/s13071-018-2667-1

    Examination of HlGST (a ) and HlGST2 ( b ) in selected tissues during blood-feeding. The salivary glands, midgut, and ovary were observed during blood-feeding of adult ticks by indirect immunofluorescent antibody test (IFAT) using a confocal laser scanning microscope . Antiserum against HlGST or HlGST2 was used for the primary antibody, anti-mouse IgG conjugated with Alexa 488 was used for the secondary antibody, and nuclei were visualized using DAPI. Normal mouse serum was used for a control. Abbreviations : Salivary glands (A, acinus; D, salivary ducts); Midgut (E, enterocytes; L, lumen); Ovary (O, oocyte; Pd, pedicel; Ow, ovarian wall). Arrows show the positive fluorescence of GST. Scale-bars : 20 μm
    Figure Legend Snippet: Examination of HlGST (a ) and HlGST2 ( b ) in selected tissues during blood-feeding. The salivary glands, midgut, and ovary were observed during blood-feeding of adult ticks by indirect immunofluorescent antibody test (IFAT) using a confocal laser scanning microscope . Antiserum against HlGST or HlGST2 was used for the primary antibody, anti-mouse IgG conjugated with Alexa 488 was used for the secondary antibody, and nuclei were visualized using DAPI. Normal mouse serum was used for a control. Abbreviations : Salivary glands (A, acinus; D, salivary ducts); Midgut (E, enterocytes; L, lumen); Ovary (O, oocyte; Pd, pedicel; Ow, ovarian wall). Arrows show the positive fluorescence of GST. Scale-bars : 20 μm

    Techniques Used: Laser-Scanning Microscopy, Fluorescence

    Localization of GSTs in tissues of partially fed adult ticks. Immunofluorescent antibody test (IFAT) was used to determine the localization of the GSTs in the different tissues of ticks. Antiserum against HlGST or HlGST2 was used for the primary antibody, anti-mouse IgG conjugated with Alexa 488 was used for the secondary antibody, and nuclei were visualized using DAPI. Normal mouse serum was used for a control. The tissues were visualized using confocal microscope. Abbreviations : Salivary glands (A, acinus; D, salivary ducts); Midgut (E, enterocytes; L, lumen; Fat bodies (T, tracheal complex; Fb, fat body cells); Ovary (O, oocyte; Pd, pedicel; Ow, ovarian wall). Arrows show positive GST fluorescence. Scale-bars : 20 μm
    Figure Legend Snippet: Localization of GSTs in tissues of partially fed adult ticks. Immunofluorescent antibody test (IFAT) was used to determine the localization of the GSTs in the different tissues of ticks. Antiserum against HlGST or HlGST2 was used for the primary antibody, anti-mouse IgG conjugated with Alexa 488 was used for the secondary antibody, and nuclei were visualized using DAPI. Normal mouse serum was used for a control. The tissues were visualized using confocal microscope. Abbreviations : Salivary glands (A, acinus; D, salivary ducts); Midgut (E, enterocytes; L, lumen; Fat bodies (T, tracheal complex; Fb, fat body cells); Ovary (O, oocyte; Pd, pedicel; Ow, ovarian wall). Arrows show positive GST fluorescence. Scale-bars : 20 μm

    Techniques Used: Microscopy, Fluorescence

    11) Product Images from "C-Terminal Amino Acids 471-507 of Avian Hepatitis E Virus Capsid Protein Are Crucial for Binding to Avian and Human Cells"

    Article Title: C-Terminal Amino Acids 471-507 of Avian Hepatitis E Virus Capsid Protein Are Crucial for Binding to Avian and Human Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0153723

    Indirect immunofluorescence staining to investigate binding to LMH cells. Cells were incubated with 500nM recombinant proteins, each, for 1h and fixed, then stained with anti-Xpress antibodies, followed by Alexa Fluor 488-conjugated anti-mouse IgG. Cells were co-stained with DAPI (blue) and Whole Cell Stain (red), afterwards imaged using (a) widefield fluorescence or (b) confocal microscopy. PBS was used as negative control. Arrows indicate the location of proteins binding to the surface of LMH cells. Bar, 20μm.
    Figure Legend Snippet: Indirect immunofluorescence staining to investigate binding to LMH cells. Cells were incubated with 500nM recombinant proteins, each, for 1h and fixed, then stained with anti-Xpress antibodies, followed by Alexa Fluor 488-conjugated anti-mouse IgG. Cells were co-stained with DAPI (blue) and Whole Cell Stain (red), afterwards imaged using (a) widefield fluorescence or (b) confocal microscopy. PBS was used as negative control. Arrows indicate the location of proteins binding to the surface of LMH cells. Bar, 20μm.

    Techniques Used: Immunofluorescence, Staining, Binding Assay, Incubation, Recombinant, Fluorescence, Confocal Microscopy, Negative Control

    12) Product Images from "Identification of Nonstructural Protein 8 as the N-Terminus of the RNA-Dependent RNA Polymerase of Porcine Reproductive and Respiratory Syndrome Virus"

    Article Title: Identification of Nonstructural Protein 8 as the N-Terminus of the RNA-Dependent RNA Polymerase of Porcine Reproductive and Respiratory Syndrome Virus

    Journal: Virologica Sinica

    doi: 10.1007/s12250-018-0054-x

    Preparation of rabbit polyclonal antibodies to PRRSV nsp8. ( A ) Analysis of the expression and purification of recombinant GST-nsp8 protein by 12% SDS-PAGE gel. ( B ) Western blot of the expression of GST-nsp8 by anti-GST mAb. ( C ) Immunofluorescence detection of HA-nsp8 in transfected MARC-145 cells by anti-GST-nsp8 serum. MARC-145 cells on coverslips in six well plates were transfected with the plasmid pHA-nsp8 (middle) or the vector pCMV-HA (right). At 18–24 h post transfection, the cells were fixed, permeablized and stained with proper antibodies to the HA epitope or to GST-nsp8, followed by Alexa Fluor 488-conjugated secondary antibodies. The cell nuclei were stained with Hoechst (blue) and examined by confocal microscopy. ( D ) Detection of nsp8 expression in PRRSV infected MARC-145 cells. MARC-145 cells grown on coverslips were either mock infected with DMEM or infected with HP-PRRSV strain JXwn06 at an MOI of 0.1. At 24 h after infection, the cells were fixed and stained with rabbit pre-immune serum or anti-GST-nsp8 serum and examined by confocal microscopy.
    Figure Legend Snippet: Preparation of rabbit polyclonal antibodies to PRRSV nsp8. ( A ) Analysis of the expression and purification of recombinant GST-nsp8 protein by 12% SDS-PAGE gel. ( B ) Western blot of the expression of GST-nsp8 by anti-GST mAb. ( C ) Immunofluorescence detection of HA-nsp8 in transfected MARC-145 cells by anti-GST-nsp8 serum. MARC-145 cells on coverslips in six well plates were transfected with the plasmid pHA-nsp8 (middle) or the vector pCMV-HA (right). At 18–24 h post transfection, the cells were fixed, permeablized and stained with proper antibodies to the HA epitope or to GST-nsp8, followed by Alexa Fluor 488-conjugated secondary antibodies. The cell nuclei were stained with Hoechst (blue) and examined by confocal microscopy. ( D ) Detection of nsp8 expression in PRRSV infected MARC-145 cells. MARC-145 cells grown on coverslips were either mock infected with DMEM or infected with HP-PRRSV strain JXwn06 at an MOI of 0.1. At 24 h after infection, the cells were fixed and stained with rabbit pre-immune serum or anti-GST-nsp8 serum and examined by confocal microscopy.

    Techniques Used: Expressing, Purification, Recombinant, SDS Page, Western Blot, Immunofluorescence, Transfection, Plasmid Preparation, Staining, Confocal Microscopy, Infection

    13) Product Images from "Enterohemorrhagic Escherichia coli Hemolysin Employs Outer Membrane Vesicles to Target Mitochondria and Cause Endothelial and Epithelial Apoptosis"

    Article Title: Enterohemorrhagic Escherichia coli Hemolysin Employs Outer Membrane Vesicles to Target Mitochondria and Cause Endothelial and Epithelial Apoptosis

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003797

    EHEC-Hly separates from OMVs during intracellular trafficking. ( A ) HBMEC and Caco-2 cells were incubated with TA50 or 8033 OMVs for the times indicated and analyzed using CLSM. OMVs were stained using mouse anti- E. coli LPS antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG (green), and EHEC-Hly (EHly) was stained using rabbit anti-EHEC-Hly antibody and Cy3-conjugated goat anti-rabbit IgG (red). Nuclei were stained with DRAQ5 (blue). Pictures were taken and processed as described in the legend to Figure 4 . Colocalized red and green signals appear in yellow (examples are indicated by white arrows). Red arrows indicate examples of red signal of EHEC-Hly dissociating from OMVs during time. The percentages of colocalization between OMVs and EHEC-Hly were calculated using BioImageXD6 colocalization tool and are indicated by white numbers (averages from at least five different samples). ( B ) HBMEC and Caco-2 cells were incubated for 24 h with EHEC-Hly-free OMVs from strains TA51 or 8033c and stained as described in panel A. ( C ) HBMEC and Caco-2 cells were incubated for 24 h with 20 mM TRIS-HCl (OMV buffer) instead of OMVs and stained for OMVs and EHEC-Hly as described in panel A or stained with secondary antibodies in the absence of primary antibodies. Scale bars in all panels are 10 µm.
    Figure Legend Snippet: EHEC-Hly separates from OMVs during intracellular trafficking. ( A ) HBMEC and Caco-2 cells were incubated with TA50 or 8033 OMVs for the times indicated and analyzed using CLSM. OMVs were stained using mouse anti- E. coli LPS antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG (green), and EHEC-Hly (EHly) was stained using rabbit anti-EHEC-Hly antibody and Cy3-conjugated goat anti-rabbit IgG (red). Nuclei were stained with DRAQ5 (blue). Pictures were taken and processed as described in the legend to Figure 4 . Colocalized red and green signals appear in yellow (examples are indicated by white arrows). Red arrows indicate examples of red signal of EHEC-Hly dissociating from OMVs during time. The percentages of colocalization between OMVs and EHEC-Hly were calculated using BioImageXD6 colocalization tool and are indicated by white numbers (averages from at least five different samples). ( B ) HBMEC and Caco-2 cells were incubated for 24 h with EHEC-Hly-free OMVs from strains TA51 or 8033c and stained as described in panel A. ( C ) HBMEC and Caco-2 cells were incubated for 24 h with 20 mM TRIS-HCl (OMV buffer) instead of OMVs and stained for OMVs and EHEC-Hly as described in panel A or stained with secondary antibodies in the absence of primary antibodies. Scale bars in all panels are 10 µm.

    Techniques Used: Incubation, Confocal Laser Scanning Microscopy, Staining

    Releasing of EHEC-Hly from lysosomes leads to a transient loss of lysosomal function. ( A, B ) HBMEC and Caco-2 cells were incubated with EHEC-Hly-containing (TA50 or 8033) or EHEC-Hly-free (TA51 or 8033c) OMVs for 8 h ( A ) and 24 h ( B ). OMVs were stained with rabbit anti- E. coli LPS antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG (green), lysosomes with Lysotracker Red DND-99 (red) and nuclei with DRAQ5 (blue). ( C, D ) HBMEC and Caco-2 cells were incubated with TA50 or 8033 OMVs ( C ) or with TA51 or 8033c OMVs ( D ) for 8 h and 24 h. EHEC-Hly (EHly) was stained with rabbit anti-EHEC-Hly antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG (green), lysosomes with Lysotracker Red DND-99 (red), and nuclei with DRAQ5 (blue). Pictures were taken and processed as described in the legend to Figure 4 . Colocalized red and green signals appear in yellow (examples in panels A and B are depicted by arrows). White numbers indicate the percentages of OMVs or EHEC-Hly colocalized with Lysotracker Red DND-99-positive lysosomes (averages from at least five different samples) calculated using the BioImageXD6 colocalization tool. Scale bars are 10 µm. The pictures shown in panel D (8 h of incubation) are also representative of 24 h (no EHEC-Hly was detected in cells treated with EHEC-Hly-free OMVs at any of these time points).
    Figure Legend Snippet: Releasing of EHEC-Hly from lysosomes leads to a transient loss of lysosomal function. ( A, B ) HBMEC and Caco-2 cells were incubated with EHEC-Hly-containing (TA50 or 8033) or EHEC-Hly-free (TA51 or 8033c) OMVs for 8 h ( A ) and 24 h ( B ). OMVs were stained with rabbit anti- E. coli LPS antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG (green), lysosomes with Lysotracker Red DND-99 (red) and nuclei with DRAQ5 (blue). ( C, D ) HBMEC and Caco-2 cells were incubated with TA50 or 8033 OMVs ( C ) or with TA51 or 8033c OMVs ( D ) for 8 h and 24 h. EHEC-Hly (EHly) was stained with rabbit anti-EHEC-Hly antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG (green), lysosomes with Lysotracker Red DND-99 (red), and nuclei with DRAQ5 (blue). Pictures were taken and processed as described in the legend to Figure 4 . Colocalized red and green signals appear in yellow (examples in panels A and B are depicted by arrows). White numbers indicate the percentages of OMVs or EHEC-Hly colocalized with Lysotracker Red DND-99-positive lysosomes (averages from at least five different samples) calculated using the BioImageXD6 colocalization tool. Scale bars are 10 µm. The pictures shown in panel D (8 h of incubation) are also representative of 24 h (no EHEC-Hly was detected in cells treated with EHEC-Hly-free OMVs at any of these time points).

    Techniques Used: Incubation, Staining

    EHEC-Hly colocalizes with mitochondria. HBMEC and Caco-2 cells were incubated with EHEC-Hly-containing OMVs from strains TA50 and 8033 or with EHEC-Hly-free OMVs from strains TA51 and 8033c (controls) or with 20 mM TRIS-HCl buffer in lieu of OMVs for 24 h. EHEC-Hly (EHly) was stained with anti-EHEC-Hly antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG (green) and mitochondria (Mito) were stained with MitoTracker Orange CMTMRos (red). DNA was stained with DRAQ5 (blue). Pictures were taken using a laser-scanning microscope (LSM 510 META microscope, equipped with a Plan-Apochromat 63x/1.4 oil immersion objective). All three fluorescence images were merged (left panels; colocalized red and green signals appear in yellow and examples are depicted by arrows) and single fluorescence channels are shown in the right panels. Pictures consisted of one optical section of a z-series with a pinhole of 1 airy unit. Scale bars are 10 µm. Note that mitotracker signals in cells treated with EHEC-Hly-containing OMVs (TA50, 8033) are slightly diffuse compared to those in cells treated with EHEC-Hly-free OMVs (TA51, 8033c) and in OMV-untreated cells, likely because of reduction of the mitochondrial transmembrane potential induced by EHEC-Hly at this time (see Figure 11E, 11F ).
    Figure Legend Snippet: EHEC-Hly colocalizes with mitochondria. HBMEC and Caco-2 cells were incubated with EHEC-Hly-containing OMVs from strains TA50 and 8033 or with EHEC-Hly-free OMVs from strains TA51 and 8033c (controls) or with 20 mM TRIS-HCl buffer in lieu of OMVs for 24 h. EHEC-Hly (EHly) was stained with anti-EHEC-Hly antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG (green) and mitochondria (Mito) were stained with MitoTracker Orange CMTMRos (red). DNA was stained with DRAQ5 (blue). Pictures were taken using a laser-scanning microscope (LSM 510 META microscope, equipped with a Plan-Apochromat 63x/1.4 oil immersion objective). All three fluorescence images were merged (left panels; colocalized red and green signals appear in yellow and examples are depicted by arrows) and single fluorescence channels are shown in the right panels. Pictures consisted of one optical section of a z-series with a pinhole of 1 airy unit. Scale bars are 10 µm. Note that mitotracker signals in cells treated with EHEC-Hly-containing OMVs (TA50, 8033) are slightly diffuse compared to those in cells treated with EHEC-Hly-free OMVs (TA51, 8033c) and in OMV-untreated cells, likely because of reduction of the mitochondrial transmembrane potential induced by EHEC-Hly at this time (see Figure 11E, 11F ).

    Techniques Used: Incubation, Staining, Laser-Scanning Microscopy, Microscopy, Fluorescence

    Bafilomycin A1 inhibits translocation of EHEC-Hly from lysosomes to mitochondria. ( A ) HBMEC and Caco-2 cells either pretreated with bafilomycin A1 (BafA1+) (100 nM, 1 h) or BafA1-untreated (BafA1-) were incubated with TA50 or 8033 OMVs or without OMVs for 24 h. Lysosomal and mitochondrial fractions were isolated and analyzed for OMVs and EHEC-Hly using immunoblot with anti-OmpA and anti-EHEC-Hly antibody, respectively. Efficiency of BafA1 treatment was verified using immunoblot with anti-LC3B antibody which detects an increased amount of processed LC3B-II in the presence of BafA1. The sizes of immunoreactive bands are indicated along the right side of Caco-2 cell blots. ( B ) BafA1-pretreated HBMEC and Caco-2 cells were incubated with TA50 or 8033 OMVs (or with control EHEC-Hly-free TA51 or 8033c OMVs) for 24 h and analysed using CLSM. Lysosomes were stained with anti-CD63 antibody and Cy3-conjugated goat anti-mouse IgG (red), EHEC-Hly (EHly) with anti-EHEC-Hly antibody and Alexa Fluor 488-conjugated IgG (green), and nuclei with DRAQ5 (blue). Pictures were taken and processed as described in the legend to Figure 4 . Colocalized red and green signals appear in yellow (examples indicated by arrows). The percentages of colocalizations of EHEC-Hly with CD63-positive compartments were calculated using the BioImageXD6 colocalization tool and are shown (averages from at least five different samples) by white numbers in images in panel B and graphically in panel ( C ). Scale bars are 10 µM. ( D ) TA50 and 8033 OMVs were treated (1 h, 37°C) with TRIS-HCl buffer with pH ranging from 8.0 to 2.0; samples were ultracentrifuged and the pellets (P) (containing OMV-associated EHEC-Hly) and supernatants (S) (containing EHEC-Hly that had separated from OMVs) were analyzed for EHEC-Hly using immunoblotting. EHEC-Hly signals in P and S fractions were quantified densitometrically and the percentage of EHEC-Hly present in the P and S fraction at each particular pH was calculated from the total EHEC-Hly signal.
    Figure Legend Snippet: Bafilomycin A1 inhibits translocation of EHEC-Hly from lysosomes to mitochondria. ( A ) HBMEC and Caco-2 cells either pretreated with bafilomycin A1 (BafA1+) (100 nM, 1 h) or BafA1-untreated (BafA1-) were incubated with TA50 or 8033 OMVs or without OMVs for 24 h. Lysosomal and mitochondrial fractions were isolated and analyzed for OMVs and EHEC-Hly using immunoblot with anti-OmpA and anti-EHEC-Hly antibody, respectively. Efficiency of BafA1 treatment was verified using immunoblot with anti-LC3B antibody which detects an increased amount of processed LC3B-II in the presence of BafA1. The sizes of immunoreactive bands are indicated along the right side of Caco-2 cell blots. ( B ) BafA1-pretreated HBMEC and Caco-2 cells were incubated with TA50 or 8033 OMVs (or with control EHEC-Hly-free TA51 or 8033c OMVs) for 24 h and analysed using CLSM. Lysosomes were stained with anti-CD63 antibody and Cy3-conjugated goat anti-mouse IgG (red), EHEC-Hly (EHly) with anti-EHEC-Hly antibody and Alexa Fluor 488-conjugated IgG (green), and nuclei with DRAQ5 (blue). Pictures were taken and processed as described in the legend to Figure 4 . Colocalized red and green signals appear in yellow (examples indicated by arrows). The percentages of colocalizations of EHEC-Hly with CD63-positive compartments were calculated using the BioImageXD6 colocalization tool and are shown (averages from at least five different samples) by white numbers in images in panel B and graphically in panel ( C ). Scale bars are 10 µM. ( D ) TA50 and 8033 OMVs were treated (1 h, 37°C) with TRIS-HCl buffer with pH ranging from 8.0 to 2.0; samples were ultracentrifuged and the pellets (P) (containing OMV-associated EHEC-Hly) and supernatants (S) (containing EHEC-Hly that had separated from OMVs) were analyzed for EHEC-Hly using immunoblotting. EHEC-Hly signals in P and S fractions were quantified densitometrically and the percentage of EHEC-Hly present in the P and S fraction at each particular pH was calculated from the total EHEC-Hly signal.

    Techniques Used: Translocation Assay, Incubation, Isolation, Confocal Laser Scanning Microscopy, Staining

    Colocalization of OMVs and EHEC-Hly with endo-lysosomal compartments detected with anti-CD63 antibody. ( A, B ) HBMEC and Caco-2 cells were incubated with EHEC-Hly-containing (TA50 or 8033) or EHEC-Hly-free (TA51 or 8033c) OMVs for 8 h ( A ) and 24 h ( B ). OMVs were stained with rabbit anti- E. coli LPS antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG (green), lysosomes with mouse anti-CD63 antibody and Cy3-conjugated goat anti-mouse IgG (red), and nuclei with DRAQ5 (blue). ( C, D ) HBMEC and Caco-2 cells were incubated with EHEC-Hly-containing (TA50 or 8033) ( C ) or EHEC-Hly-free (TA51 or 8033c) OMVs ( D ) for 8 h and 24 h and stained as described above except that in lieu of OMVs, EHEC-Hly (EHly) was detected with rabbit anti-EHEC-Hly antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). Pictures were taken and processed as described in the legend to Figure 4 . Colocalized red and green signals appear in yellow (examples indicated by arrows). White numbers indicate the percentages of OMVs ( A, B ) and EHEC-Hly ( C ) colocalized with CD63-positive compartments (averages from at least five different samples) calculated using the BioImageXD6 colocalization tool. Scale bars are 10 µm. The images in panel D (8 h of incubation) are also representative of 24 h (no EHEC-Hly was detected in cells treated with EHEC-Hly-free OMVs at any of these time points).
    Figure Legend Snippet: Colocalization of OMVs and EHEC-Hly with endo-lysosomal compartments detected with anti-CD63 antibody. ( A, B ) HBMEC and Caco-2 cells were incubated with EHEC-Hly-containing (TA50 or 8033) or EHEC-Hly-free (TA51 or 8033c) OMVs for 8 h ( A ) and 24 h ( B ). OMVs were stained with rabbit anti- E. coli LPS antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG (green), lysosomes with mouse anti-CD63 antibody and Cy3-conjugated goat anti-mouse IgG (red), and nuclei with DRAQ5 (blue). ( C, D ) HBMEC and Caco-2 cells were incubated with EHEC-Hly-containing (TA50 or 8033) ( C ) or EHEC-Hly-free (TA51 or 8033c) OMVs ( D ) for 8 h and 24 h and stained as described above except that in lieu of OMVs, EHEC-Hly (EHly) was detected with rabbit anti-EHEC-Hly antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). Pictures were taken and processed as described in the legend to Figure 4 . Colocalized red and green signals appear in yellow (examples indicated by arrows). White numbers indicate the percentages of OMVs ( A, B ) and EHEC-Hly ( C ) colocalized with CD63-positive compartments (averages from at least five different samples) calculated using the BioImageXD6 colocalization tool. Scale bars are 10 µm. The images in panel D (8 h of incubation) are also representative of 24 h (no EHEC-Hly was detected in cells treated with EHEC-Hly-free OMVs at any of these time points).

    Techniques Used: Incubation, Staining

    14) Product Images from "Identification of Nonstructural Protein 8 as the N-Terminus of the RNA-Dependent RNA Polymerase of Porcine Reproductive and Respiratory Syndrome Virus"

    Article Title: Identification of Nonstructural Protein 8 as the N-Terminus of the RNA-Dependent RNA Polymerase of Porcine Reproductive and Respiratory Syndrome Virus

    Journal: Virologica Sinica

    doi: 10.1007/s12250-018-0054-x

    Preparation of rabbit polyclonal antibodies to PRRSV nsp8. ( A ) Analysis of the expression and purification of recombinant GST-nsp8 protein by 12% SDS-PAGE gel. ( B ) Western blot of the expression of GST-nsp8 by anti-GST mAb. ( C ) Immunofluorescence detection of HA-nsp8 in transfected MARC-145 cells by anti-GST-nsp8 serum. MARC-145 cells on coverslips in six well plates were transfected with the plasmid pHA-nsp8 (middle) or the vector pCMV-HA (right). At 18–24 h post transfection, the cells were fixed, permeablized and stained with proper antibodies to the HA epitope or to GST-nsp8, followed by Alexa Fluor 488-conjugated secondary antibodies. The cell nuclei were stained with Hoechst (blue) and examined by confocal microscopy. ( D ) Detection of nsp8 expression in PRRSV infected MARC-145 cells. MARC-145 cells grown on coverslips were either mock infected with DMEM or infected with HP-PRRSV strain JXwn06 at an MOI of 0.1. At 24 h after infection, the cells were fixed and stained with rabbit pre-immune serum or anti-GST-nsp8 serum and examined by confocal microscopy.
    Figure Legend Snippet: Preparation of rabbit polyclonal antibodies to PRRSV nsp8. ( A ) Analysis of the expression and purification of recombinant GST-nsp8 protein by 12% SDS-PAGE gel. ( B ) Western blot of the expression of GST-nsp8 by anti-GST mAb. ( C ) Immunofluorescence detection of HA-nsp8 in transfected MARC-145 cells by anti-GST-nsp8 serum. MARC-145 cells on coverslips in six well plates were transfected with the plasmid pHA-nsp8 (middle) or the vector pCMV-HA (right). At 18–24 h post transfection, the cells were fixed, permeablized and stained with proper antibodies to the HA epitope or to GST-nsp8, followed by Alexa Fluor 488-conjugated secondary antibodies. The cell nuclei were stained with Hoechst (blue) and examined by confocal microscopy. ( D ) Detection of nsp8 expression in PRRSV infected MARC-145 cells. MARC-145 cells grown on coverslips were either mock infected with DMEM or infected with HP-PRRSV strain JXwn06 at an MOI of 0.1. At 24 h after infection, the cells were fixed and stained with rabbit pre-immune serum or anti-GST-nsp8 serum and examined by confocal microscopy.

    Techniques Used: Expressing, Purification, Recombinant, SDS Page, Western Blot, Immunofluorescence, Transfection, Plasmid Preparation, Staining, Confocal Microscopy, Infection

    15) Product Images from "Antiviral activity of lambda-carrageenan against influenza viruses and severe acute respiratory syndrome coronavirus 2"

    Article Title: Antiviral activity of lambda-carrageenan against influenza viruses and severe acute respiratory syndrome coronavirus 2

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-80896-9

    Effect of λ-CGN on the influenza A virus entry. ( A ) HA inhibition assay. Two-fold serially diluted PR8 (from 2 3  to 2 11 ) in PBS was incubated with an equal volume of PBS or twofold increasing concentrations of λ-CGN for 20 min. HA titer in each combination was determined at 30 min after addition of 0.5% chicken RBC. HA titers are marked on the right side of the panel. ( B , C ) Confocal microscopy. MDCK cells were infected with PR8 (MOI, 5) in the absence (Mock) or presence of either λ-CGN or p-KG03 at a concentration of 10 μg/ml. At 4 h post-infection in the absence of CHX ( B ) or at 2.5 h in the presence of 10 μg/ml CHX ( C ), viral NP was detected with an anti-NP antibody and an Alex Fluor 488-conjugated goat anti-mouse secondary antibody (green). Cell nuclei were counterstained with DAPI (blue). Original magnification, 400×. The images were analyzed using ZEN blue software 3.1 ( www.zeiss.com ).
    Figure Legend Snippet: Effect of λ-CGN on the influenza A virus entry. ( A ) HA inhibition assay. Two-fold serially diluted PR8 (from 2 3 to 2 11 ) in PBS was incubated with an equal volume of PBS or twofold increasing concentrations of λ-CGN for 20 min. HA titer in each combination was determined at 30 min after addition of 0.5% chicken RBC. HA titers are marked on the right side of the panel. ( B , C ) Confocal microscopy. MDCK cells were infected with PR8 (MOI, 5) in the absence (Mock) or presence of either λ-CGN or p-KG03 at a concentration of 10 μg/ml. At 4 h post-infection in the absence of CHX ( B ) or at 2.5 h in the presence of 10 μg/ml CHX ( C ), viral NP was detected with an anti-NP antibody and an Alex Fluor 488-conjugated goat anti-mouse secondary antibody (green). Cell nuclei were counterstained with DAPI (blue). Original magnification, 400×. The images were analyzed using ZEN blue software 3.1 ( www.zeiss.com ).

    Techniques Used: Inhibition, Incubation, Confocal Microscopy, Infection, Concentration Assay, Software

    Image-based antiviral analysis of λ-CGN against SARS-CoV-2. ( A ) Vero cells seeded in 96-well plates were infected with SARS-CoV-2 at an MOI of 0.02, either alone or in the presence of increasing concentrations of λ-CGN (upper panel) or RDV (lower panel; a control). On day 2 post-infection, cells were fixed and permeabilized prior to immunostaining with an anti-SARS-CoV-2 spike antibody and an Alexa Fluor 488-conjugated goat anti-mouse IgG (green). Cell nuclei were counterstained with DAPI to estimate cell viability (blue). Images were captured with a 20× objective lens fitted to an automated fluorescence microscope by using the Harmony High-Content Imaging and Analysis software 3.5.2 ( www.perkinelmer.com ). ( B ) The number of fluorescent green and blue spots was counted to calculate antiviral activity (red circles) and cell viability (black squares), respectively, at each concentration of the compounds. The viability of mock-infected cells were fixed as 100%, while the antiviral activity in virus-infected cells or mock-infected cells was fixed as 0 and 100%, respectively. Data are expressed as the mean ± S.D. from three independent experiments. The graphs were created using GraphPad Prism 8.3.1 ( www.graphpad.com ).
    Figure Legend Snippet: Image-based antiviral analysis of λ-CGN against SARS-CoV-2. ( A ) Vero cells seeded in 96-well plates were infected with SARS-CoV-2 at an MOI of 0.02, either alone or in the presence of increasing concentrations of λ-CGN (upper panel) or RDV (lower panel; a control). On day 2 post-infection, cells were fixed and permeabilized prior to immunostaining with an anti-SARS-CoV-2 spike antibody and an Alexa Fluor 488-conjugated goat anti-mouse IgG (green). Cell nuclei were counterstained with DAPI to estimate cell viability (blue). Images were captured with a 20× objective lens fitted to an automated fluorescence microscope by using the Harmony High-Content Imaging and Analysis software 3.5.2 ( www.perkinelmer.com ). ( B ) The number of fluorescent green and blue spots was counted to calculate antiviral activity (red circles) and cell viability (black squares), respectively, at each concentration of the compounds. The viability of mock-infected cells were fixed as 100%, while the antiviral activity in virus-infected cells or mock-infected cells was fixed as 0 and 100%, respectively. Data are expressed as the mean ± S.D. from three independent experiments. The graphs were created using GraphPad Prism 8.3.1 ( www.graphpad.com ).

    Techniques Used: Infection, Immunostaining, Fluorescence, Microscopy, Imaging, Software, Activity Assay, Concentration Assay

    16) Product Images from "Rhesus Rhadinovirus Infection of Rhesus Fibroblasts Occurs through Clathrin-Mediated Endocytosis ▿"

    Article Title: Rhesus Rhadinovirus Infection of Rhesus Fibroblasts Occurs through Clathrin-Mediated Endocytosis ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01429-10

    RRV particles are colocalized with markers of clathrin-mediated endocytosis during entry into RFs. (A to D) Detection of colocalization of RRV-RFP particles with Alexa Fluor 488-transferrin (A), clathrin (B), and EEA1 (C), but not with Alexa Fluor 488-CTB
    Figure Legend Snippet: RRV particles are colocalized with markers of clathrin-mediated endocytosis during entry into RFs. (A to D) Detection of colocalization of RRV-RFP particles with Alexa Fluor 488-transferrin (A), clathrin (B), and EEA1 (C), but not with Alexa Fluor 488-CTB

    Techniques Used: CtB Assay

    17) Product Images from "Generation of cleidocranial dysplasia-specific human induced pluripotent stem cells in completely serum-, feeder-, and integration-free culture"

    Article Title: Generation of cleidocranial dysplasia-specific human induced pluripotent stem cells in completely serum-, feeder-, and integration-free culture

    Journal: In Vitro Cellular & Developmental Biology. Animal

    doi: 10.1007/s11626-015-9968-x

    Differentiation ability of CCD-iPSCs derived from DPCs in serum-, feeder- and integration-free defined culture conditions. ( A ) Differentiation was performed using embryoid body formation, and the differentiated iPSCs (CCD-SeV-iPS1 clone8) were fixed and reacted with antibodies. Shown were immunocytochemistry of Nestin, βIII-tubulin, MAP-2, α-smooth muscle actin ( α-SMA ), and α-fetoprotein ( AFP ). Binding of these antibodies was visualized with Alexa Fluor® 488-conjugated secondary antibodies ( green ). Nucleuses were stained with DAPI. (passage 27) Bar indicates 100 μm. ( B ) Teratoma formation of CCD-iPSCs in the defined culture conditions in SCID mice. Teratomas were generated in SCID mice (CB17/Icr- Prkdc scid /CrlCrlj) from CCD-SeV-iPS1 (clone3 at passage 28). Histological analysis with H-E staining demonstrated that teratomas formed from CCD-iPSCs cultured in hESF9-based conditions contained derivatives of all three germ layers. Scale bars represent 200 μm. ( C ) Karyotype analysis of CCD-SeV-iPS1 clone3 at passage 48 generated in hESF9 had a normal diploid 46, XX karyotype. ( D ) Sequencing result of dental pulp cell of CCD donor ( CCD-DPC ) and generated CCD-iPS cell ( CCD-iPS1 ) demonstrated heterozygous mutation in RUNX2 . The DPC carried a missense mutation (647G → A) which was responsible for arginine to glutamine substitution [R225Q]. WT : wild type (WT) allele pattern in RUNX2.
    Figure Legend Snippet: Differentiation ability of CCD-iPSCs derived from DPCs in serum-, feeder- and integration-free defined culture conditions. ( A ) Differentiation was performed using embryoid body formation, and the differentiated iPSCs (CCD-SeV-iPS1 clone8) were fixed and reacted with antibodies. Shown were immunocytochemistry of Nestin, βIII-tubulin, MAP-2, α-smooth muscle actin ( α-SMA ), and α-fetoprotein ( AFP ). Binding of these antibodies was visualized with Alexa Fluor® 488-conjugated secondary antibodies ( green ). Nucleuses were stained with DAPI. (passage 27) Bar indicates 100 μm. ( B ) Teratoma formation of CCD-iPSCs in the defined culture conditions in SCID mice. Teratomas were generated in SCID mice (CB17/Icr- Prkdc scid /CrlCrlj) from CCD-SeV-iPS1 (clone3 at passage 28). Histological analysis with H-E staining demonstrated that teratomas formed from CCD-iPSCs cultured in hESF9-based conditions contained derivatives of all three germ layers. Scale bars represent 200 μm. ( C ) Karyotype analysis of CCD-SeV-iPS1 clone3 at passage 48 generated in hESF9 had a normal diploid 46, XX karyotype. ( D ) Sequencing result of dental pulp cell of CCD donor ( CCD-DPC ) and generated CCD-iPS cell ( CCD-iPS1 ) demonstrated heterozygous mutation in RUNX2 . The DPC carried a missense mutation (647G → A) which was responsible for arginine to glutamine substitution [R225Q]. WT : wild type (WT) allele pattern in RUNX2.

    Techniques Used: Derivative Assay, Immunocytochemistry, Binding Assay, Staining, Mouse Assay, Generated, Cell Culture, Sequencing, Mutagenesis

    Characterization of CCD-iPSCs generated in serum-, feeder-, and integration-free culture conditions. ( A ) Expression of ES cell marker genes in iPSCs derived from CCD-DPCs. We used primers that only amplified the endogenous genes (Takahashi et al. 2007 ; Nishimura et al. 2007 ; Yamasaki et al. 2014 ). ( #1 ) CCD-DPC: passage 1 = before infection ( #2 ) CCD-SeV-iPS1 clone3: passage 11 = serum-free condition ( #3 ) CCD-SeV-iPS1 clone4: passage 12 = serum-free condition ( #4 ) CCD-SeV-iPS1 clone7: passage 13 = serum-free condition ( #5 ) CCD-SeV-iPS1 clone8: passage 12 = serum-free condition ( #6 ): CCD-SeV-iPS1 clone1: passage 7 = serum-supplemented condition CCD-DPC-derived iPS cells were designated CCD-SeV-iPS. Full-length blots are shown in Supplementary Figure S7 . ( B ) ALP activity of generated CCD-iPSCs The ALP activity was detected (CCD-SeV-iPS1 clone3 at passage 68). Bars indicate 200 μm. ( C ) Immunocytochemistry of pluripotency marker proteins CCD-SeV-iPS1-clone3 grown under hESF9-based culture conditions for 42 passages were fixed and reacted with antibodies (Oct4, Tra-1-60 and Tra-1-81, SSEA-4, SeVdp). Binding of these antibodies was visualized with Alexa Fluor® 488-conjugated secondary antibodies ( green ). Nuclei were stained with DAPI ( blue ). Scale bars represent 100 μm.
    Figure Legend Snippet: Characterization of CCD-iPSCs generated in serum-, feeder-, and integration-free culture conditions. ( A ) Expression of ES cell marker genes in iPSCs derived from CCD-DPCs. We used primers that only amplified the endogenous genes (Takahashi et al. 2007 ; Nishimura et al. 2007 ; Yamasaki et al. 2014 ). ( #1 ) CCD-DPC: passage 1 = before infection ( #2 ) CCD-SeV-iPS1 clone3: passage 11 = serum-free condition ( #3 ) CCD-SeV-iPS1 clone4: passage 12 = serum-free condition ( #4 ) CCD-SeV-iPS1 clone7: passage 13 = serum-free condition ( #5 ) CCD-SeV-iPS1 clone8: passage 12 = serum-free condition ( #6 ): CCD-SeV-iPS1 clone1: passage 7 = serum-supplemented condition CCD-DPC-derived iPS cells were designated CCD-SeV-iPS. Full-length blots are shown in Supplementary Figure S7 . ( B ) ALP activity of generated CCD-iPSCs The ALP activity was detected (CCD-SeV-iPS1 clone3 at passage 68). Bars indicate 200 μm. ( C ) Immunocytochemistry of pluripotency marker proteins CCD-SeV-iPS1-clone3 grown under hESF9-based culture conditions for 42 passages were fixed and reacted with antibodies (Oct4, Tra-1-60 and Tra-1-81, SSEA-4, SeVdp). Binding of these antibodies was visualized with Alexa Fluor® 488-conjugated secondary antibodies ( green ). Nuclei were stained with DAPI ( blue ). Scale bars represent 100 μm.

    Techniques Used: Generated, Expressing, Marker, Derivative Assay, Amplification, Infection, ALP Assay, Activity Assay, Immunocytochemistry, Binding Assay, Staining

    18) Product Images from "Outer Membrane Vesicles From Probiotic and Commensal Escherichia coli Activate NOD1-Mediated Immune Responses in Intestinal Epithelial Cells"

    Article Title: Outer Membrane Vesicles From Probiotic and Commensal Escherichia coli Activate NOD1-Mediated Immune Responses in Intestinal Epithelial Cells

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.00498

    NOD1 colocalizes with EcN and ECOR12 OMVs. HT-29 cells were incubated with EcN or ECOR12 OMVs (10 μg) for 1 h and analyzed using laser scanning confocal spectral microscope. NOD1 was stained using anti-NOD1 polyclonal antibody and Alexa Fluor 633-conjugated goat anti-rabbit IgG (red). OMVs were stained using anti- Escherichia coli LPS mouse monoclonal antibody followed by Alexa Fluor 488-conjugated goat anti-mouse IgG (green). Images are representative of three independent biological experiments. Colocalization of the red and green signals was confirmed by histogram analysis of the fluorescence intensities along the yellow lines. Analysis was performed by laser scanning confocal spectral microscope with 63x oil immersion objective lens, and images were captured with a Nikon color camera (8 bit). Scale bar: 10 μm.
    Figure Legend Snippet: NOD1 colocalizes with EcN and ECOR12 OMVs. HT-29 cells were incubated with EcN or ECOR12 OMVs (10 μg) for 1 h and analyzed using laser scanning confocal spectral microscope. NOD1 was stained using anti-NOD1 polyclonal antibody and Alexa Fluor 633-conjugated goat anti-rabbit IgG (red). OMVs were stained using anti- Escherichia coli LPS mouse monoclonal antibody followed by Alexa Fluor 488-conjugated goat anti-mouse IgG (green). Images are representative of three independent biological experiments. Colocalization of the red and green signals was confirmed by histogram analysis of the fluorescence intensities along the yellow lines. Analysis was performed by laser scanning confocal spectral microscope with 63x oil immersion objective lens, and images were captured with a Nikon color camera (8 bit). Scale bar: 10 μm.

    Techniques Used: Incubation, Microscopy, Staining, Fluorescence

    19) Product Images from "Analysis of Bovine Leukemia Virus Gag Membrane Targeting and Late Domain Function"

    Article Title: Analysis of Bovine Leukemia Virus Gag Membrane Targeting and Late Domain Function

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.16.8485-8493.2002

    The amino-terminal glycine is required for efficient VLP production and Pr Gag membrane targeting. (A) VLP production. pGag-HA and mutants were stably transfected into COS-1 cells. Cell and virion lysates were analyzed by Western blot using an anti-HA antibody. The Western blots were subjected to quantitative fluorochemical analysis as described in the text. This experiment was repeated three times, with representative results shown. (B) Cellular distribution of Pr Gag . Cells stably transfected with VLP constructs were grown on coverslips, fixed, and incubated with an anti-HA Ig followed by incubation with Alexa Fluor 488-conjugated anti-mouse Ig. Images were collected using a confocal microscope.
    Figure Legend Snippet: The amino-terminal glycine is required for efficient VLP production and Pr Gag membrane targeting. (A) VLP production. pGag-HA and mutants were stably transfected into COS-1 cells. Cell and virion lysates were analyzed by Western blot using an anti-HA antibody. The Western blots were subjected to quantitative fluorochemical analysis as described in the text. This experiment was repeated three times, with representative results shown. (B) Cellular distribution of Pr Gag . Cells stably transfected with VLP constructs were grown on coverslips, fixed, and incubated with an anti-HA Ig followed by incubation with Alexa Fluor 488-conjugated anti-mouse Ig. Images were collected using a confocal microscope.

    Techniques Used: Stable Transfection, Transfection, Western Blot, Construct, Incubation, Microscopy

    20) Product Images from "Preventing abnormal NF-κB activation and autoimmunity by Otub1-mediated p100 stabilization"

    Article Title: Preventing abnormal NF-κB activation and autoimmunity by Otub1-mediated p100 stabilization

    Journal: Cell Research

    doi: 10.1038/s41422-019-0174-3

    Otub1 -BKO mice develop lupus-like autoimmunity. a ELISA of basal concentrations of autoantibodies reacting against double-stranded DNA (anti-dsDNA) or nuclear antigen (ANA) in the serum of unimmunized wild-type or Otub1-BKO mice (10 months old). b Immunofluorescent microscopy of IgG deposits in kidney sections from young (8 weeks) or older (10 months) unimmunized wild-type and Otub1-BKO mice. Sections were stained with DAPI (blue) and Alexa Fluor 488 anti-mouse IgG (green). Scale bar, 100 μm. Quantification of the intensity of signal was performed by ImageJ software. Data are presented as a representative plot (left panel) and summary graph (right panel). c A representative spleen image (left) and a summary graph of spleen weight (right, each circle represents a mouse) of wild-type and Otub1 -BKO mice that were either not treated (NT) or injected (i.p.) with CD4 T cells from BM12 mice. d , e Flow cytometric analysis of Fas + GL-7 + germinal center (GC) B cells and B220 − CD138 + plasma cells in wild-type and Otub1 -BKO mice that were either not treated (NT) or injected (i.p.) with CD4 T cells from BM12 mice for the indicated time periods. Data are presented as a representative plot (left) and summary graph based on multiple recipient mice (right). f ELISA of autoantibodies reacting against double-stranded DNA (anti-dsDNA) and nuclear antigen (ANA) in serum from wild-type and Otub1 -BKO mice injected (i.p.) with CD4 T cells from BM12 mice for the indicated time periods. g Immunofluorescent microscopy of IgG deposits in kidney sections from wild-type and Otub1- BKO mice injected (i.p.) with CD4 T cells from BM12 mice for the indicated time periods. Sections were stained with DAPI (blue) and FITC anti-mouse IgG (green). Scale bar, 100 μm. Quantification of the fluorescent signal was performed by ImageJ software. Data are presented as a representative plot (left panel) and summary graph (right panel). Data are representative of three independent experiments. P values are determined by two-tailed unpaired t -test. * P
    Figure Legend Snippet: Otub1 -BKO mice develop lupus-like autoimmunity. a ELISA of basal concentrations of autoantibodies reacting against double-stranded DNA (anti-dsDNA) or nuclear antigen (ANA) in the serum of unimmunized wild-type or Otub1-BKO mice (10 months old). b Immunofluorescent microscopy of IgG deposits in kidney sections from young (8 weeks) or older (10 months) unimmunized wild-type and Otub1-BKO mice. Sections were stained with DAPI (blue) and Alexa Fluor 488 anti-mouse IgG (green). Scale bar, 100 μm. Quantification of the intensity of signal was performed by ImageJ software. Data are presented as a representative plot (left panel) and summary graph (right panel). c A representative spleen image (left) and a summary graph of spleen weight (right, each circle represents a mouse) of wild-type and Otub1 -BKO mice that were either not treated (NT) or injected (i.p.) with CD4 T cells from BM12 mice. d , e Flow cytometric analysis of Fas + GL-7 + germinal center (GC) B cells and B220 − CD138 + plasma cells in wild-type and Otub1 -BKO mice that were either not treated (NT) or injected (i.p.) with CD4 T cells from BM12 mice for the indicated time periods. Data are presented as a representative plot (left) and summary graph based on multiple recipient mice (right). f ELISA of autoantibodies reacting against double-stranded DNA (anti-dsDNA) and nuclear antigen (ANA) in serum from wild-type and Otub1 -BKO mice injected (i.p.) with CD4 T cells from BM12 mice for the indicated time periods. g Immunofluorescent microscopy of IgG deposits in kidney sections from wild-type and Otub1- BKO mice injected (i.p.) with CD4 T cells from BM12 mice for the indicated time periods. Sections were stained with DAPI (blue) and FITC anti-mouse IgG (green). Scale bar, 100 μm. Quantification of the fluorescent signal was performed by ImageJ software. Data are presented as a representative plot (left panel) and summary graph (right panel). Data are representative of three independent experiments. P values are determined by two-tailed unpaired t -test. * P

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Microscopy, Staining, Software, Injection, Two Tailed Test

    21) Product Images from "2',3'-Cyclic Nucleotide 3'-Phosphodiesterases Inhibit Hepatitis B Virus Replication"

    Article Title: 2',3'-Cyclic Nucleotide 3'-Phosphodiesterases Inhibit Hepatitis B Virus Replication

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0080769

    Native expression of CNP in human hepatoma cell lines. (A) The expression of the CNP proteins was studied by immunofluorescence microscopy using the anti-CNP polyclonal rabbit antibody and Alexa Fluor 488-anti-rabbit IgG. The nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). (B) Huh7 and HepG2 cells were cultured without or with 300, 1000 IU/ml of IFNα-2b for 2 days. Cells were harvested and the cell lysates were determined by Western blot analysis. Actin served as a loading control (protein panel). (C) For CNP mRNA quantitative analysis, cDNA pools were synthesized from the 1 μg of total RNA of cells and diluted cDNA was amplified by quantitative real-time PCR. Actin was served as reverse transcription and PCR control. The mRNA% presents the ratio of values of total CNP (tCNP) to that of naive HepG2 cells (n=3).
    Figure Legend Snippet: Native expression of CNP in human hepatoma cell lines. (A) The expression of the CNP proteins was studied by immunofluorescence microscopy using the anti-CNP polyclonal rabbit antibody and Alexa Fluor 488-anti-rabbit IgG. The nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). (B) Huh7 and HepG2 cells were cultured without or with 300, 1000 IU/ml of IFNα-2b for 2 days. Cells were harvested and the cell lysates were determined by Western blot analysis. Actin served as a loading control (protein panel). (C) For CNP mRNA quantitative analysis, cDNA pools were synthesized from the 1 μg of total RNA of cells and diluted cDNA was amplified by quantitative real-time PCR. Actin was served as reverse transcription and PCR control. The mRNA% presents the ratio of values of total CNP (tCNP) to that of naive HepG2 cells (n=3).

    Techniques Used: Expressing, Immunofluorescence, Microscopy, Cell Culture, Western Blot, Synthesized, Amplification, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    Subcellular location of CNP isoforms in Huh7 cells. (A) Huh7 cells were transfected with pCDNA5-CNP1 or pCDNA5-CNP2 and harvested at day 2 post transfection. The expression of CNP and tubulin was revealed by immunofluorescence microscopy using the anti-CNP polyclonal rabbit antibody and anti-tublin monoclonal mice antibody, the secondary antibodies were Alexa Fluor 594-conjugated goat anti-rabbit IgG Alexa Fluor 488-conjugated goat anti-mouse or rabbit IgG. The nuclei were counterstained with DAPI. Mitochondria were stained using Mitocapture Tm kit and indicated by red color. White arrow indicates a CNP1-transfected Huh7 cell undergone a morphological change. (B) Huh7 ells in 12-well plate were transfected with 2 μg of plasmids pCDNA5-CNP1 or pCDNA5-CNP2. Cell lysates were determined by Western blot analysis. Actin served as a loading control.
    Figure Legend Snippet: Subcellular location of CNP isoforms in Huh7 cells. (A) Huh7 cells were transfected with pCDNA5-CNP1 or pCDNA5-CNP2 and harvested at day 2 post transfection. The expression of CNP and tubulin was revealed by immunofluorescence microscopy using the anti-CNP polyclonal rabbit antibody and anti-tublin monoclonal mice antibody, the secondary antibodies were Alexa Fluor 594-conjugated goat anti-rabbit IgG Alexa Fluor 488-conjugated goat anti-mouse or rabbit IgG. The nuclei were counterstained with DAPI. Mitochondria were stained using Mitocapture Tm kit and indicated by red color. White arrow indicates a CNP1-transfected Huh7 cell undergone a morphological change. (B) Huh7 ells in 12-well plate were transfected with 2 μg of plasmids pCDNA5-CNP1 or pCDNA5-CNP2. Cell lysates were determined by Western blot analysis. Actin served as a loading control.

    Techniques Used: Transfection, Expressing, Immunofluorescence, Microscopy, Mouse Assay, Staining, Western Blot

    22) Product Images from "Characterization and expression analysis of a newly identified glutathione S-transferase of the hard tick Haemaphysalis longicornis during blood-feeding"

    Article Title: Characterization and expression analysis of a newly identified glutathione S-transferase of the hard tick Haemaphysalis longicornis during blood-feeding

    Journal: Parasites & Vectors

    doi: 10.1186/s13071-018-2667-1

    Examination of HlGST (a ) and HlGST2 ( b ) in selected tissues during blood-feeding. The salivary glands, midgut, and ovary were observed during blood-feeding of adult ticks by indirect immunofluorescent antibody test (IFAT) using a confocal laser scanning microscope . Antiserum against HlGST or HlGST2 was used for the primary antibody, anti-mouse IgG conjugated with Alexa 488 was used for the secondary antibody, and nuclei were visualized using DAPI. Normal mouse serum was used for a control. Abbreviations : Salivary glands (A, acinus; D, salivary ducts); Midgut (E, enterocytes; L, lumen); Ovary (O, oocyte; Pd, pedicel; Ow, ovarian wall). Arrows show the positive fluorescence of GST. Scale-bars : 20 μm
    Figure Legend Snippet: Examination of HlGST (a ) and HlGST2 ( b ) in selected tissues during blood-feeding. The salivary glands, midgut, and ovary were observed during blood-feeding of adult ticks by indirect immunofluorescent antibody test (IFAT) using a confocal laser scanning microscope . Antiserum against HlGST or HlGST2 was used for the primary antibody, anti-mouse IgG conjugated with Alexa 488 was used for the secondary antibody, and nuclei were visualized using DAPI. Normal mouse serum was used for a control. Abbreviations : Salivary glands (A, acinus; D, salivary ducts); Midgut (E, enterocytes; L, lumen); Ovary (O, oocyte; Pd, pedicel; Ow, ovarian wall). Arrows show the positive fluorescence of GST. Scale-bars : 20 μm

    Techniques Used: Laser-Scanning Microscopy, Fluorescence

    Localization of GSTs in tissues of partially fed adult ticks. Immunofluorescent antibody test (IFAT) was used to determine the localization of the GSTs in the different tissues of ticks. Antiserum against HlGST or HlGST2 was used for the primary antibody, anti-mouse IgG conjugated with Alexa 488 was used for the secondary antibody, and nuclei were visualized using DAPI. Normal mouse serum was used for a control. The tissues were visualized using confocal microscope. Abbreviations : Salivary glands (A, acinus; D, salivary ducts); Midgut (E, enterocytes; L, lumen; Fat bodies (T, tracheal complex; Fb, fat body cells); Ovary (O, oocyte; Pd, pedicel; Ow, ovarian wall). Arrows show positive GST fluorescence. Scale-bars : 20 μm
    Figure Legend Snippet: Localization of GSTs in tissues of partially fed adult ticks. Immunofluorescent antibody test (IFAT) was used to determine the localization of the GSTs in the different tissues of ticks. Antiserum against HlGST or HlGST2 was used for the primary antibody, anti-mouse IgG conjugated with Alexa 488 was used for the secondary antibody, and nuclei were visualized using DAPI. Normal mouse serum was used for a control. The tissues were visualized using confocal microscope. Abbreviations : Salivary glands (A, acinus; D, salivary ducts); Midgut (E, enterocytes; L, lumen; Fat bodies (T, tracheal complex; Fb, fat body cells); Ovary (O, oocyte; Pd, pedicel; Ow, ovarian wall). Arrows show positive GST fluorescence. Scale-bars : 20 μm

    Techniques Used: Microscopy, Fluorescence

    23) Product Images from "Octa-Arginine Mediated Delivery of Wild-Type Lnk Protein Inhibits TPO-Induced M-MOK Megakaryoblastic Leukemic Cell Growth by Promoting Apoptosis"

    Article Title: Octa-Arginine Mediated Delivery of Wild-Type Lnk Protein Inhibits TPO-Induced M-MOK Megakaryoblastic Leukemic Cell Growth by Promoting Apoptosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0023640

    WT Lnk R8 binds endogenous Jak2 and impairs TPO-mediated Jak-Stat and MAPK activation. (A) M-MOK cells treated with WT or mutant Lnk R8 were either stimulated or not stimulated with TPO. Cells were collected and thoroughly washed before lysed. Lysates were immunoprecipitated with anti-Jak2 or control IgG antibody and analyzed by Western blotting with anti-Jak2, anti-pentaHis or anti-c-mpl antibodies. IgG control lane represents immunoprecipitation from WT Lnk R8-treated sample (with TPO stimulation) using control IgG antibody. (B) Protein-treated M-MOK cells were stimulated with TPO for 30 minutes. Cells were fixed and probed with anti-pentaHis and anti-Jak2 antibodies. Secondary Alexa 488 and Alexa 594 conjugated antibodies were used to detect primary antibodies (Green: Lnk R8, Red: Jak2). Co-localization of Lnk R8 and Jak2 (yellow) were shown in merged pictures. (C) Protein treated-M-MOK cells were stimulated with 10 ng/ml of TPO for 0, 10, 30 minutes. Cells were harvested and lysates were subjected to Western blot analyses. Phosphorylation and total protein levels of Jak2, Stat5 and MAPK were detected using antibodies as described in Materials and Methods .
    Figure Legend Snippet: WT Lnk R8 binds endogenous Jak2 and impairs TPO-mediated Jak-Stat and MAPK activation. (A) M-MOK cells treated with WT or mutant Lnk R8 were either stimulated or not stimulated with TPO. Cells were collected and thoroughly washed before lysed. Lysates were immunoprecipitated with anti-Jak2 or control IgG antibody and analyzed by Western blotting with anti-Jak2, anti-pentaHis or anti-c-mpl antibodies. IgG control lane represents immunoprecipitation from WT Lnk R8-treated sample (with TPO stimulation) using control IgG antibody. (B) Protein-treated M-MOK cells were stimulated with TPO for 30 minutes. Cells were fixed and probed with anti-pentaHis and anti-Jak2 antibodies. Secondary Alexa 488 and Alexa 594 conjugated antibodies were used to detect primary antibodies (Green: Lnk R8, Red: Jak2). Co-localization of Lnk R8 and Jak2 (yellow) were shown in merged pictures. (C) Protein treated-M-MOK cells were stimulated with 10 ng/ml of TPO for 0, 10, 30 minutes. Cells were harvested and lysates were subjected to Western blot analyses. Phosphorylation and total protein levels of Jak2, Stat5 and MAPK were detected using antibodies as described in Materials and Methods .

    Techniques Used: Activation Assay, Mutagenesis, Immunoprecipitation, Western Blot

    24) Product Images from "Osteoclasts Modulate Bone Erosion in Cholesteatoma via RANKL Signaling"

    Article Title: Osteoclasts Modulate Bone Erosion in Cholesteatoma via RANKL Signaling

    Journal: JARO: Journal of the Association for Research in Otolaryngology

    doi: 10.1007/s10162-019-00727-1

    Fibroblasts in the cholesteatoma perimatrix express RANKL. a Expression of RANKL mRNA in cholesteatoma, corrected for GAPDH mRNA, is significantly higher than in control skin ( n = 9). The asterisk indicates a statistically significant difference. b DAB staining for RANKL in the cholesteatoma perimatrix, counterstained with hematoxylin (scale bars 50 μm). c Immunofluorescent staining of the cholesteatoma perimatrix. Co-localization of vimentin and RANKL immunofluorescence shows that fibroblasts in the perimatrix express RANKL. RANKL was labeled with Alexa Fluor® 555, and vimentin was labeled with Alexa Fluor® 488. Nuclei were stained with Hoechst® 33342 (scale bars: 10 μm)
    Figure Legend Snippet: Fibroblasts in the cholesteatoma perimatrix express RANKL. a Expression of RANKL mRNA in cholesteatoma, corrected for GAPDH mRNA, is significantly higher than in control skin ( n = 9). The asterisk indicates a statistically significant difference. b DAB staining for RANKL in the cholesteatoma perimatrix, counterstained with hematoxylin (scale bars 50 μm). c Immunofluorescent staining of the cholesteatoma perimatrix. Co-localization of vimentin and RANKL immunofluorescence shows that fibroblasts in the perimatrix express RANKL. RANKL was labeled with Alexa Fluor® 555, and vimentin was labeled with Alexa Fluor® 488. Nuclei were stained with Hoechst® 33342 (scale bars: 10 μm)

    Techniques Used: Expressing, Staining, Immunofluorescence, Labeling

    25) Product Images from "Peli1 negatively regulates noncanonical NF-κB signaling to restrain systemic lupus erythematosus"

    Article Title: Peli1 negatively regulates noncanonical NF-κB signaling to restrain systemic lupus erythematosus

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03530-3

    The noncanonical NF-κB pathway is required for lupus pathology in Peli1-deficient mice. a – c Rag1-dificient mice (recipient, R) were adoptively transferred with WT or KO B cells (donor, D), and then immunized with BM12 CD4 + T cell. The IgG deposits in kidney were determined by staining with Alexa Fluor 488-labeled anti-mouse IgG ( a , Scale bar, 100 μm), the serum ANA were analyzed by using the Hep-2 cell line ( b ), and the anti-dsDNA, anti-ssDNA, anti-histone IgG in serum were examined by ELISA ( c ). d Proliferation of WT and KO splenic B cells that infected with control or NIK shRNA, incubated in vitro for 72 h in the absence (NT) or presence of anti-CD40 or BAFF, then assessed by [ 3 H]thymidine incorporation. e – g Rag1-dificient mice were adoptively transferred with WT or KO B cells that infected with control or NIK shRNA, and then immunized with BM12 CD4 + T cell. The kidney IgG deposition were visualized with Alexa Fluor 488-labeled anti-mouse IgG ( e , Scale bar, 100 μm), the serum ANA were analyzed by using the Hep-2 cell line ( f , Scale bar, 50 μm.), and the anti-dsDNA, anti-ssDNA, anti-histone IgG in serum were examined by ELISA ( g ). Ctrl, control. Data are shown as the mean ± SEM based on three independent experiments. Two-tailed Student’s t- tests were performed. * P
    Figure Legend Snippet: The noncanonical NF-κB pathway is required for lupus pathology in Peli1-deficient mice. a – c Rag1-dificient mice (recipient, R) were adoptively transferred with WT or KO B cells (donor, D), and then immunized with BM12 CD4 + T cell. The IgG deposits in kidney were determined by staining with Alexa Fluor 488-labeled anti-mouse IgG ( a , Scale bar, 100 μm), the serum ANA were analyzed by using the Hep-2 cell line ( b ), and the anti-dsDNA, anti-ssDNA, anti-histone IgG in serum were examined by ELISA ( c ). d Proliferation of WT and KO splenic B cells that infected with control or NIK shRNA, incubated in vitro for 72 h in the absence (NT) or presence of anti-CD40 or BAFF, then assessed by [ 3 H]thymidine incorporation. e – g Rag1-dificient mice were adoptively transferred with WT or KO B cells that infected with control or NIK shRNA, and then immunized with BM12 CD4 + T cell. The kidney IgG deposition were visualized with Alexa Fluor 488-labeled anti-mouse IgG ( e , Scale bar, 100 μm), the serum ANA were analyzed by using the Hep-2 cell line ( f , Scale bar, 50 μm.), and the anti-dsDNA, anti-ssDNA, anti-histone IgG in serum were examined by ELISA ( g ). Ctrl, control. Data are shown as the mean ± SEM based on three independent experiments. Two-tailed Student’s t- tests were performed. * P

    Techniques Used: Mouse Assay, Staining, Labeling, Enzyme-linked Immunosorbent Assay, Infection, shRNA, Incubation, In Vitro, Two Tailed Test

    Overexpression of Peli1 prevents lupus-like disease. a Immunoblot analysis of p52, p100, NIK, Peli1, Peli1ΔC and HSP60 in Peli1 -knockdown M12 cells that reconstituted with empty vector (EV), WT full-length Peli1 or Peli1ΔC that stimulated with anti-CD40 (αCD40) at the indicated time points. b Analysis of Lys48 ubiquitination of NIK in Peli1 -knockdown M12 cells reconstituted with EV, WT Peli1, or Peli1ΔC that stimulated with anti-CD40 (αCD40) for 4 h in the presence of MG132. c LISA of NP-specific IgM, IgG2a, IgG2b and IgG3 in the serum of Rag1 −/− mice that transferred with WT T cells plus KO B cells reconstituted with EV, WT Peli1 or Peli1ΔC, and then immunized intraperitoneally with NP-KLH. d – h Peli1 -deficient (KO) mice were immunized with 7.5 million of BM12 CD4 + T cell to induce lupus-like disease, and then injected with pRV-GFP retrovirus encoding Peli1 or Peli1ΔC 12 h post-immunization. The IgG deposits in kidney were examined by staining with Alexa Fluor 488-labeled anti-mouse IgG ( d ). Scale bar, 100 μm. The anti-dsDNA, anti-ssDNA, anti-histone IgG in serum were determined by ELISA ( e ). The percentages of CD19 − CD138 + plasma cells, PD-1 + CXCR5 + Tfh cells ( f , g ) and infected B220 + B cells ( h ) in spleens were assessed by flow cytometry. Data are shown as the mean ± SEM based on three independent experiments. Two-tailed Student’s t- tests were performed. * P
    Figure Legend Snippet: Overexpression of Peli1 prevents lupus-like disease. a Immunoblot analysis of p52, p100, NIK, Peli1, Peli1ΔC and HSP60 in Peli1 -knockdown M12 cells that reconstituted with empty vector (EV), WT full-length Peli1 or Peli1ΔC that stimulated with anti-CD40 (αCD40) at the indicated time points. b Analysis of Lys48 ubiquitination of NIK in Peli1 -knockdown M12 cells reconstituted with EV, WT Peli1, or Peli1ΔC that stimulated with anti-CD40 (αCD40) for 4 h in the presence of MG132. c LISA of NP-specific IgM, IgG2a, IgG2b and IgG3 in the serum of Rag1 −/− mice that transferred with WT T cells plus KO B cells reconstituted with EV, WT Peli1 or Peli1ΔC, and then immunized intraperitoneally with NP-KLH. d – h Peli1 -deficient (KO) mice were immunized with 7.5 million of BM12 CD4 + T cell to induce lupus-like disease, and then injected with pRV-GFP retrovirus encoding Peli1 or Peli1ΔC 12 h post-immunization. The IgG deposits in kidney were examined by staining with Alexa Fluor 488-labeled anti-mouse IgG ( d ). Scale bar, 100 μm. The anti-dsDNA, anti-ssDNA, anti-histone IgG in serum were determined by ELISA ( e ). The percentages of CD19 − CD138 + plasma cells, PD-1 + CXCR5 + Tfh cells ( f , g ) and infected B220 + B cells ( h ) in spleens were assessed by flow cytometry. Data are shown as the mean ± SEM based on three independent experiments. Two-tailed Student’s t- tests were performed. * P

    Techniques Used: Over Expression, Plasmid Preparation, Mouse Assay, Injection, Staining, Labeling, Enzyme-linked Immunosorbent Assay, Infection, Flow Cytometry, Cytometry, Two Tailed Test

    Peli1 deficiency aggravates the induction of lupus-like disease. a WT and Peli1 -deficient (KO) mice were intraperitoneally injected with 7.5 million of CD4 + T cell from C57BL/6 mice (control) or from BM12 mice. Representative immunofluorescent images showing IgG deposits in kidney by staining with Alexa Fluor 488-labeled anti-mouse IgG. Scale bar, 100 μm. b Flow cytometric analysis of the percentages of CD19 − CD138 + plasma cells and Fas + GL-7 + germinal center (GC) B cells in WT and KO immunized mice as described in ( a ). Data are presented as a representative plot (left panel) and summary graph (right panel). c Distinct anti-nuclear antibody (ANA) staining patterns of the Hep-2 cell line with serum from WT and KO mice 4 weeks after immunization as described in ( a ). Scale bar, 50 μm. d Enzyme-linked immunosorbent assay (ELISA) of anti-dsDNA, anti-ssDNA, and anti-histone IgG in serum from WT and KO immunized mice as described in ( a ) at the indicate time point. e Immunoblot showing NIK and p52 protein levels in splenic B cells from control and BM12 CD4 + T cells immunized WT and KO mice as described in ( a ). Data are shown as the mean ± SEM based on three independent experiments. Two-tailed Student’s t- tests were performed. * P
    Figure Legend Snippet: Peli1 deficiency aggravates the induction of lupus-like disease. a WT and Peli1 -deficient (KO) mice were intraperitoneally injected with 7.5 million of CD4 + T cell from C57BL/6 mice (control) or from BM12 mice. Representative immunofluorescent images showing IgG deposits in kidney by staining with Alexa Fluor 488-labeled anti-mouse IgG. Scale bar, 100 μm. b Flow cytometric analysis of the percentages of CD19 − CD138 + plasma cells and Fas + GL-7 + germinal center (GC) B cells in WT and KO immunized mice as described in ( a ). Data are presented as a representative plot (left panel) and summary graph (right panel). c Distinct anti-nuclear antibody (ANA) staining patterns of the Hep-2 cell line with serum from WT and KO mice 4 weeks after immunization as described in ( a ). Scale bar, 50 μm. d Enzyme-linked immunosorbent assay (ELISA) of anti-dsDNA, anti-ssDNA, and anti-histone IgG in serum from WT and KO immunized mice as described in ( a ) at the indicate time point. e Immunoblot showing NIK and p52 protein levels in splenic B cells from control and BM12 CD4 + T cells immunized WT and KO mice as described in ( a ). Data are shown as the mean ± SEM based on three independent experiments. Two-tailed Student’s t- tests were performed. * P

    Techniques Used: Mouse Assay, Injection, Staining, Labeling, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    Peli1 deficiency specifically in B cells promotes autoimmunity in vivo. a Scheme showing how the μMT chimeric mice were constructed for immunization. b Enzyme-linked immunosorbent assay (ELISA) of NP-specific IgG1, IgG2a, IgG2b, and IgG3 in the serum of WT/μMT and Peli1-deficient (KO)/μMT chimeric mice that immunized intraperitoneally with vehicle (sham) or NP-KLH (KLH). c WT/μMT and KO/μMT chimeric mice were intraperitoneally injected with 7.5 million of CD4 + T cell from C57BL/6 mice (control) or from BM12 mice. Representative immunofluorescent images showing IgG deposits in kidney by staining with Alexa Fluor 488-labeled anti-mouse IgG. Scale bar, 100 μm. d , e Flow cytometric analysis of the percentages of Fas + GL-7 + germinal center (GC) B cells and CD19 − CD138 + plasma cells in immunized WT/μMT and KO/μMT chimeric mice as described in ( c ). Data are presented as the representative FACS plots ( d ) and summary graphs ( e ). f Distinct anti-nuclear antibody (ANA) staining patterns of the Hep-2 cell line with serum from WT/μMT and KO/μMT chimeric mice 4 weeks after immunization as described in ( c ). Ctr represents control in the right panel bar graph. Scale bar, 50 μm. g Enzyme-linked immunosorbent assay (ELISA) of anti-dsDNA, anti-ssDNA, and anti-histone IgG in serum from immunized WT/μMT and KO/μMT chimeric mice as described in ( c ) at the indicate time point. Data are shown as the mean ± SEM based on three independent experiments. Two-tailed Student’s t- tests were performed. * P
    Figure Legend Snippet: Peli1 deficiency specifically in B cells promotes autoimmunity in vivo. a Scheme showing how the μMT chimeric mice were constructed for immunization. b Enzyme-linked immunosorbent assay (ELISA) of NP-specific IgG1, IgG2a, IgG2b, and IgG3 in the serum of WT/μMT and Peli1-deficient (KO)/μMT chimeric mice that immunized intraperitoneally with vehicle (sham) or NP-KLH (KLH). c WT/μMT and KO/μMT chimeric mice were intraperitoneally injected with 7.5 million of CD4 + T cell from C57BL/6 mice (control) or from BM12 mice. Representative immunofluorescent images showing IgG deposits in kidney by staining with Alexa Fluor 488-labeled anti-mouse IgG. Scale bar, 100 μm. d , e Flow cytometric analysis of the percentages of Fas + GL-7 + germinal center (GC) B cells and CD19 − CD138 + plasma cells in immunized WT/μMT and KO/μMT chimeric mice as described in ( c ). Data are presented as the representative FACS plots ( d ) and summary graphs ( e ). f Distinct anti-nuclear antibody (ANA) staining patterns of the Hep-2 cell line with serum from WT/μMT and KO/μMT chimeric mice 4 weeks after immunization as described in ( c ). Ctr represents control in the right panel bar graph. Scale bar, 50 μm. g Enzyme-linked immunosorbent assay (ELISA) of anti-dsDNA, anti-ssDNA, and anti-histone IgG in serum from immunized WT/μMT and KO/μMT chimeric mice as described in ( c ) at the indicate time point. Data are shown as the mean ± SEM based on three independent experiments. Two-tailed Student’s t- tests were performed. * P

    Techniques Used: In Vivo, Mouse Assay, Construct, Enzyme-linked Immunosorbent Assay, Injection, Staining, Labeling, Flow Cytometry, FACS, Two Tailed Test

    26) Product Images from "Intracellular Trafficking of Clostridium perfringens Iota-Toxin b"

    Article Title: Intracellular Trafficking of Clostridium perfringens Iota-Toxin b

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00483-12

    Colocalization of Ib and endosome markers in MDCK cells. (A) MDCK cells were incubated with Ib (1 μg/ml) at 4°C for 1 h, washed, and incubated at 37°C for the period indicated. Cells were fixed, permeabilized, and stained with DAPI and antibodies to EEA1, Lamp2, Golgi 58K, and Ib. MDCK cells were transiently transfected with pGFP-Rab11, pGFP-lysosome, or pER-GFP. After 24 h of transfection, the transfected cells were incubated with Ib as described above. Cells were fixed, permeabilized, and stained with anti-Ib antibody and DAPI. Ib (red), endosome markers (green), and the nucleus (blue) were viewed with a confocal microscope. The experiments were repeated three times, and a representative result is shown. Bar, 5 μm. (B) Negative-control preparation. Cells were fixed, permeabilized, and stained with Alexa Fluor 568-conjugated anti-rabbit IgG, FITC-conjugated anti-mouse IgG, and DAPI. (C) Quantification of colocalizations. The percentage of Ib/endocytic marker colocalization represents the ratio of the number of endosomal structures stained for Ib and for endocytic marker to the total number of endosomal structures stained for endocytic marker. The percentage of colocalization was determined for each cell, and the results represent the average ± standard error of the mean (SEM) for several cells ( n > 10) obtained from at least three independent experiments.
    Figure Legend Snippet: Colocalization of Ib and endosome markers in MDCK cells. (A) MDCK cells were incubated with Ib (1 μg/ml) at 4°C for 1 h, washed, and incubated at 37°C for the period indicated. Cells were fixed, permeabilized, and stained with DAPI and antibodies to EEA1, Lamp2, Golgi 58K, and Ib. MDCK cells were transiently transfected with pGFP-Rab11, pGFP-lysosome, or pER-GFP. After 24 h of transfection, the transfected cells were incubated with Ib as described above. Cells were fixed, permeabilized, and stained with anti-Ib antibody and DAPI. Ib (red), endosome markers (green), and the nucleus (blue) were viewed with a confocal microscope. The experiments were repeated three times, and a representative result is shown. Bar, 5 μm. (B) Negative-control preparation. Cells were fixed, permeabilized, and stained with Alexa Fluor 568-conjugated anti-rabbit IgG, FITC-conjugated anti-mouse IgG, and DAPI. (C) Quantification of colocalizations. The percentage of Ib/endocytic marker colocalization represents the ratio of the number of endosomal structures stained for Ib and for endocytic marker to the total number of endosomal structures stained for endocytic marker. The percentage of colocalization was determined for each cell, and the results represent the average ± standard error of the mean (SEM) for several cells ( n > 10) obtained from at least three independent experiments.

    Techniques Used: Incubation, Staining, Transfection, Microscopy, Negative Control, Marker

    27) Product Images from "Endosomal sorting of VAMP3 is regulated by PI4K2A"

    Article Title: Endosomal sorting of VAMP3 is regulated by PI4K2A

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.148809

    TfR delivery to the ERC and rate of recycling are reduced upon PI4K2A depletion. After treatment of COS-7 cells with control siRNA or siRNA directed against PI4K2A, cells were serum-starved for 30 min, then incubated with Alexa-Fluor-488-conjugated transferrin for 10 min and either fixed (A,B) or chased in complete medium for 30 min prior to fixation in 4% paraformaldehyde (C,D). PI4K2A knockdown (A, right panel) impedes delivery of transferrin to the ERC in comparison to the control siRNA-treated cells (A, left panels). (B) Appearance of transferrin in the perinuclear ERC in cells treated with siRNA oligonucleotides, shown in A, or transfected with TeNT. The ratio of transferrin fluorescence in the ERC (F ERC ) and total fluorescence (F total ) was determined as described in the Materials and Methods. Shown are ratios of mean+s.e.m. fluorescence values ( n = 50). Statistical significance was determined by one-way ANOVA analysis. * P
    Figure Legend Snippet: TfR delivery to the ERC and rate of recycling are reduced upon PI4K2A depletion. After treatment of COS-7 cells with control siRNA or siRNA directed against PI4K2A, cells were serum-starved for 30 min, then incubated with Alexa-Fluor-488-conjugated transferrin for 10 min and either fixed (A,B) or chased in complete medium for 30 min prior to fixation in 4% paraformaldehyde (C,D). PI4K2A knockdown (A, right panel) impedes delivery of transferrin to the ERC in comparison to the control siRNA-treated cells (A, left panels). (B) Appearance of transferrin in the perinuclear ERC in cells treated with siRNA oligonucleotides, shown in A, or transfected with TeNT. The ratio of transferrin fluorescence in the ERC (F ERC ) and total fluorescence (F total ) was determined as described in the Materials and Methods. Shown are ratios of mean+s.e.m. fluorescence values ( n = 50). Statistical significance was determined by one-way ANOVA analysis. * P

    Techniques Used: Incubation, Transfection, Fluorescence

    28) Product Images from "Arginase-II Induces Vascular Smooth Muscle Cell Senescence and Apoptosis Through p66Shc and p53 Independently of Its l-Arginine Ureahydrolase Activity: Implications for Atherosclerotic Plaque Vulnerability"

    Article Title: Arginase-II Induces Vascular Smooth Muscle Cell Senescence and Apoptosis Through p66Shc and p53 Independently of Its l-Arginine Ureahydrolase Activity: Implications for Atherosclerotic Plaque Vulnerability

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

    doi: 10.1161/JAHA.113.000096

    Ablation of Arg‐II in atherosclerosis‐prone ApoE −/− mice reduces levels of Arg‐II, S6K1, p66Shc, p53‐S15, and p53, as well as PCNA in VSMCs along with decreased apoptotic VSMCs in atherosclerotic plaque. ApoE −/− Arg‐II +/+ and ApoE −/− Arg‐II −/− mice were fed a high‐fat diet for 10 weeks. A, AR‐cryosections (7 μm) were subjected to immunofluorescence costaining of Arg‐II, S6K1, p66Shc, p53‐S15 and p53, and PCNA with anti‐α‐smooth muscle actin antibody (α‐SMA) for VSMCs. Mouse anti‐α‐SMA (green) was used for costaining with rabbit antibodies against Arg‐II, p66Shc, p53‐S15, and PCNA (red), whereas rabbit anti‐α‐SMA (red) was used for costaining with mouse anti‐p53 and ‐S6K1 (green). Alexa Fluor 488–conjugated goat anti‐mouse IgG (green) and Alexa Fluor 594–conjugated goat anti‐rabbit F(ab) 2 (red) were used as secondary antibodies. All sections were counterstained with DAPI. Representative images of individual staining and merged images are shown (n=4). B, Representative images showing apoptotic cells detected by TUNEL staining (red) in the plaques in aortic roots of ApoE −/− Arg‐II +/+ and ApoE −/− Arg‐II −/− mice. All sections were stained for VSMCs with anti‐α‐SMA (green) followed by counterstaining with DAPI for nuclei (blue). The merged images are also shown (n=4). The white arrows indicate apoptotic VSMCs. Scale bars=0.2 mm. Arg‐II indicates arginase‐II; ApoE, apolipoprotein E; S6K1, ribosomal protein S6 kinase 1; VSMC, vascular smooth muscle cell; PCNA, proliferating cell nuclear antigen; DAPI, 4′6‐diamidino‐2‐phenyl‐indole, dihydrochloride; TUNEL, terminal dUTP nick end‐labeling.
    Figure Legend Snippet: Ablation of Arg‐II in atherosclerosis‐prone ApoE −/− mice reduces levels of Arg‐II, S6K1, p66Shc, p53‐S15, and p53, as well as PCNA in VSMCs along with decreased apoptotic VSMCs in atherosclerotic plaque. ApoE −/− Arg‐II +/+ and ApoE −/− Arg‐II −/− mice were fed a high‐fat diet for 10 weeks. A, AR‐cryosections (7 μm) were subjected to immunofluorescence costaining of Arg‐II, S6K1, p66Shc, p53‐S15 and p53, and PCNA with anti‐α‐smooth muscle actin antibody (α‐SMA) for VSMCs. Mouse anti‐α‐SMA (green) was used for costaining with rabbit antibodies against Arg‐II, p66Shc, p53‐S15, and PCNA (red), whereas rabbit anti‐α‐SMA (red) was used for costaining with mouse anti‐p53 and ‐S6K1 (green). Alexa Fluor 488–conjugated goat anti‐mouse IgG (green) and Alexa Fluor 594–conjugated goat anti‐rabbit F(ab) 2 (red) were used as secondary antibodies. All sections were counterstained with DAPI. Representative images of individual staining and merged images are shown (n=4). B, Representative images showing apoptotic cells detected by TUNEL staining (red) in the plaques in aortic roots of ApoE −/− Arg‐II +/+ and ApoE −/− Arg‐II −/− mice. All sections were stained for VSMCs with anti‐α‐SMA (green) followed by counterstaining with DAPI for nuclei (blue). The merged images are also shown (n=4). The white arrows indicate apoptotic VSMCs. Scale bars=0.2 mm. Arg‐II indicates arginase‐II; ApoE, apolipoprotein E; S6K1, ribosomal protein S6 kinase 1; VSMC, vascular smooth muscle cell; PCNA, proliferating cell nuclear antigen; DAPI, 4′6‐diamidino‐2‐phenyl‐indole, dihydrochloride; TUNEL, terminal dUTP nick end‐labeling.

    Techniques Used: Mouse Assay, Immunofluorescence, Staining, TUNEL Assay, End Labeling

    29) Product Images from "Synergy of TLR3 and 7 ligands significantly enhances function of DCs to present inactivated PRRSV antigen through TRIF/MyD88-NF-κB signaling pathway"

    Article Title: Synergy of TLR3 and 7 ligands significantly enhances function of DCs to present inactivated PRRSV antigen through TRIF/MyD88-NF-κB signaling pathway

    Journal: Scientific Reports

    doi: 10.1038/srep23977

    Phagocytosis of MoDCs treated with TLR3 and 7 ligands along with inactivated PRRSV antigen. MoDCs were incubated with poly (I: C) and/or imiquimod along with inactivated PRRSV antigen for 12 h. MoDCs were fixed and permeabilized and then stained with monoclonal antibody against PRRSV GP5. After washing with PBS, cells were incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG(H + L) for flow cytometry. ( A ) Representative flow cytometry profile of the percentages of MoDCs catching PRRSV GP5 antigen. The data presented here are results from one experiment of three flow cytometry experiments. ( B ) The statistical graph of the percentages of MoDCs catching PRRSV GP5 antigen. The data were analyzed using Flowjo7.6 software. Data represent the means ± standard deviations (error bars) of three independent experiments. Different letters (a–e) mean significant difference ( P
    Figure Legend Snippet: Phagocytosis of MoDCs treated with TLR3 and 7 ligands along with inactivated PRRSV antigen. MoDCs were incubated with poly (I: C) and/or imiquimod along with inactivated PRRSV antigen for 12 h. MoDCs were fixed and permeabilized and then stained with monoclonal antibody against PRRSV GP5. After washing with PBS, cells were incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG(H + L) for flow cytometry. ( A ) Representative flow cytometry profile of the percentages of MoDCs catching PRRSV GP5 antigen. The data presented here are results from one experiment of three flow cytometry experiments. ( B ) The statistical graph of the percentages of MoDCs catching PRRSV GP5 antigen. The data were analyzed using Flowjo7.6 software. Data represent the means ± standard deviations (error bars) of three independent experiments. Different letters (a–e) mean significant difference ( P

    Techniques Used: Incubation, Staining, Flow Cytometry, Cytometry, Software

    30) Product Images from "Modified mRNA-LNP Vaccines Confer Protection against Experimental DENV-2 Infection in Mice"

    Article Title: Modified mRNA-LNP Vaccines Confer Protection against Experimental DENV-2 Infection in Mice

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2020.07.013

    Characterization of ADE Activities and IgG Subclass Using Immune Sera Obtained from Mice Immunized with mRNA Vaccines (A) Assessing antibody-dependent enhancement of DENV-2 infection of immune sera in K562 cells. Serial dilutions of heat-inactivated mouse sera were pre-incubated with DENV-2, followed by the addition of K562 cells. Cells were harvested 48 h after infection with immune-complexes and stained with a primary antibody against E protein (4G2 antibody) and then with a secondary anti-mouse antibody labeled with Alexa Fluor 488. The percentages of infection were determined by fluorescence-activated cell sorting (FACS). (B) Sera from E80-mRNA vaccine (left) and E80-mRNA+NS1-mRNA vaccine (right) were analyzed for IgG1, IgG2a, IgG2b, and IgG3 ratio by isotype ELISA using coating antigen DENV-2 16681 virion.
    Figure Legend Snippet: Characterization of ADE Activities and IgG Subclass Using Immune Sera Obtained from Mice Immunized with mRNA Vaccines (A) Assessing antibody-dependent enhancement of DENV-2 infection of immune sera in K562 cells. Serial dilutions of heat-inactivated mouse sera were pre-incubated with DENV-2, followed by the addition of K562 cells. Cells were harvested 48 h after infection with immune-complexes and stained with a primary antibody against E protein (4G2 antibody) and then with a secondary anti-mouse antibody labeled with Alexa Fluor 488. The percentages of infection were determined by fluorescence-activated cell sorting (FACS). (B) Sera from E80-mRNA vaccine (left) and E80-mRNA+NS1-mRNA vaccine (right) were analyzed for IgG1, IgG2a, IgG2b, and IgG3 ratio by isotype ELISA using coating antigen DENV-2 16681 virion.

    Techniques Used: Mouse Assay, Infection, Incubation, Staining, Labeling, Fluorescence, FACS, Enzyme-linked Immunosorbent Assay

    31) Product Images from "Human cytomegalovirus induces apoptosis in neural stem/progenitor cells derived from induced pluripotent stem cells by generating mitochondrial dysfunction and endoplasmic reticulum stress"

    Article Title: Human cytomegalovirus induces apoptosis in neural stem/progenitor cells derived from induced pluripotent stem cells by generating mitochondrial dysfunction and endoplasmic reticulum stress

    Journal: Herpesviridae

    doi: 10.1186/2042-4280-4-2

    Differentiation of MRC-iPS-25 cells to neural stem/progenitor cells. (A) Phase-contrast images of MRC-iPS-25 cells cultured on a feeder layer of MEFs (left) and NSPC/iPSCs (right). (B) Immunofluorescence analysis of Towne-infected MRC-iPS-25 cells cultured under feeder-free conditions at 2 dpi stained with primary antibodies for pluripotent markers (Nanog or Oct-4) and HCMV IE1/IE2 proteins. Antigen proteins were detected with Alexa Fluor 488-conjugated goat anti-mouse IgG or Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody. Nuclei were stained with DAPI. (C) Immunofluorescence analysis of NSPC markers Nestin, Sox2, and Pax6 in NSPC/iPSCs. NSPC/iPSCs were fixed and reacted with anti-Nestin (green), anti-Sox2 (red), and anti-Pax6 (red) antibodies, followed by detection with secondary antibodies. Immunofluorescence signals were obtained using a fluorescence microscope IX71. Representative results from three independent experiments are shown.
    Figure Legend Snippet: Differentiation of MRC-iPS-25 cells to neural stem/progenitor cells. (A) Phase-contrast images of MRC-iPS-25 cells cultured on a feeder layer of MEFs (left) and NSPC/iPSCs (right). (B) Immunofluorescence analysis of Towne-infected MRC-iPS-25 cells cultured under feeder-free conditions at 2 dpi stained with primary antibodies for pluripotent markers (Nanog or Oct-4) and HCMV IE1/IE2 proteins. Antigen proteins were detected with Alexa Fluor 488-conjugated goat anti-mouse IgG or Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody. Nuclei were stained with DAPI. (C) Immunofluorescence analysis of NSPC markers Nestin, Sox2, and Pax6 in NSPC/iPSCs. NSPC/iPSCs were fixed and reacted with anti-Nestin (green), anti-Sox2 (red), and anti-Pax6 (red) antibodies, followed by detection with secondary antibodies. Immunofluorescence signals were obtained using a fluorescence microscope IX71. Representative results from three independent experiments are shown.

    Techniques Used: Cell Culture, Immunofluorescence, Infection, Staining, Fluorescence, Microscopy

    HCMV-induced apoptosis of NSPC/iPSCs. (A) Towne-infected NSPC/iPSCs at 6 dpi were subjected to TUNEL assay followed by immunofluorescence test with an anti-gB antibody. TUNEL-positive nuclei were stained in red. The anti-gB antibody was detected with Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (green). Nuclei were stained with DAPI. (B-D) Towne-infected NSPC/iPSCs at 3 dpi were subjected to immunofluorescence test with anti-IE1/IE2 antibody in combination with anti-cleaved caspase 3 (B) , anti-cleaved caspase 9 (C) , or anti-cytochrome c (D) antibody. Alexa Fluor 488-conjugated goat anti-mouse IgG (green) or Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody (red) was used as a secondary antibody. Nuclei were stained with DAPI. (E) Mock-infected NSPC/iPSCs were subjected to immunofluorescence test with anti-IE1/IE2 antibody in combination with anti-cleaved caspase 3 (upper panel), anti-cleaved caspase 9 (middle panel), or anti-cytochrome c (lower panel) antibody. Nuclei were stained with DAPI. Representative results from two independent experiments are shown.
    Figure Legend Snippet: HCMV-induced apoptosis of NSPC/iPSCs. (A) Towne-infected NSPC/iPSCs at 6 dpi were subjected to TUNEL assay followed by immunofluorescence test with an anti-gB antibody. TUNEL-positive nuclei were stained in red. The anti-gB antibody was detected with Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (green). Nuclei were stained with DAPI. (B-D) Towne-infected NSPC/iPSCs at 3 dpi were subjected to immunofluorescence test with anti-IE1/IE2 antibody in combination with anti-cleaved caspase 3 (B) , anti-cleaved caspase 9 (C) , or anti-cytochrome c (D) antibody. Alexa Fluor 488-conjugated goat anti-mouse IgG (green) or Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody (red) was used as a secondary antibody. Nuclei were stained with DAPI. (E) Mock-infected NSPC/iPSCs were subjected to immunofluorescence test with anti-IE1/IE2 antibody in combination with anti-cleaved caspase 3 (upper panel), anti-cleaved caspase 9 (middle panel), or anti-cytochrome c (lower panel) antibody. Nuclei were stained with DAPI. Representative results from two independent experiments are shown.

    Techniques Used: Infection, TUNEL Assay, Immunofluorescence, Staining

    32) Product Images from "Inhibition of Epstein-Barr Virus Lytic Cycle by an Ethyl Acetate Subfraction Separated from Polygonum cuspidatum Root and Its Major Component, Emodin"

    Article Title: Inhibition of Epstein-Barr Virus Lytic Cycle by an Ethyl Acetate Subfraction Separated from Polygonum cuspidatum Root and Its Major Component, Emodin

    Journal: Molecules

    doi: 10.3390/molecules19011258

    Indirect immunofluorescence analysis of inhibition of the expressions of EBV lytic genes by F3 and emodin. P3HR1 cells were pre-treated with F3 (0.8, 1.6, 3.1 and 6.3 μg/mL) ( A ) or emodin (1.1, 2.1 and 4.2 μg/mL) ( B ) before lytic induction for 24 h. Cells were then harvested and the expressions of the lytic EBV proteins were assessed by immunofluorescence. P3HR1 cells untreated with sodium butyrate (SB), F3 and emodin were used as a negative control (Latent). The following primary antibodies were used: mouse monoclonal anti-Rta, anti-Zta, and anti-EA-D antibodies. The secondary antibodies were used: Alexa Fluor 488-conjugated goat anti-mouse IgG. DNA was visualized by DAPI staining; cells were examined under a fluorescence microscope.
    Figure Legend Snippet: Indirect immunofluorescence analysis of inhibition of the expressions of EBV lytic genes by F3 and emodin. P3HR1 cells were pre-treated with F3 (0.8, 1.6, 3.1 and 6.3 μg/mL) ( A ) or emodin (1.1, 2.1 and 4.2 μg/mL) ( B ) before lytic induction for 24 h. Cells were then harvested and the expressions of the lytic EBV proteins were assessed by immunofluorescence. P3HR1 cells untreated with sodium butyrate (SB), F3 and emodin were used as a negative control (Latent). The following primary antibodies were used: mouse monoclonal anti-Rta, anti-Zta, and anti-EA-D antibodies. The secondary antibodies were used: Alexa Fluor 488-conjugated goat anti-mouse IgG. DNA was visualized by DAPI staining; cells were examined under a fluorescence microscope.

    Techniques Used: Immunofluorescence, Inhibition, Negative Control, Staining, Fluorescence, Microscopy

    Inhibition effects of F3 and emodin on the number of cells expressing EBV lytic proteins by flow cytometric analysis. Various concentrations of F3 (0.8, 1.6, 3.1, 6.3 and 12.5 μg/mL) or emodin (Em) (1.1, 2.1, 4.2, 8.5 μg/mL) pre-treated P3HR1 cells, then SB (3 mM) co-treated cells for EBV induction. At 24 h after treatment, cells were stained with primary mouse monoclonal antibodies (anti-Rta, anti-Zta or anti-EA-D) and Alexa Fluor 488-conjugated goat anti-mouse IgG and then analyzed by flow cytometry. In histogram, M2 represented fluorescence stained cells ( A , B ). The number of cells expressing EBV lytic proteins was quantified by averaging the results from three independent experiments ( C , D ). The data were presented as means with standard deviation.
    Figure Legend Snippet: Inhibition effects of F3 and emodin on the number of cells expressing EBV lytic proteins by flow cytometric analysis. Various concentrations of F3 (0.8, 1.6, 3.1, 6.3 and 12.5 μg/mL) or emodin (Em) (1.1, 2.1, 4.2, 8.5 μg/mL) pre-treated P3HR1 cells, then SB (3 mM) co-treated cells for EBV induction. At 24 h after treatment, cells were stained with primary mouse monoclonal antibodies (anti-Rta, anti-Zta or anti-EA-D) and Alexa Fluor 488-conjugated goat anti-mouse IgG and then analyzed by flow cytometry. In histogram, M2 represented fluorescence stained cells ( A , B ). The number of cells expressing EBV lytic proteins was quantified by averaging the results from three independent experiments ( C , D ). The data were presented as means with standard deviation.

    Techniques Used: Inhibition, Expressing, Flow Cytometry, Staining, Cytometry, Fluorescence, Standard Deviation

    33) Product Images from "Maternal alloimmune IgG causes anti-glomerular basement membrane disease in perinatal transgenic mice that express human laminin α5."

    Article Title: Maternal alloimmune IgG causes anti-glomerular basement membrane disease in perinatal transgenic mice that express human laminin α5.

    Journal: Kidney international

    doi: 10.1016/j.kint.2019.06.014

    Human laminin α5 is expressed in GBMs of hu LAMA5 transgenic mice. Representative sections of kidneys from littermates born from hu LAMA5 female mice crossed with wildtype males. When cryosections from hu LAMA5 transgenic pups (upper left) and wildtype mice (lower left) were immunolabeled with mouse anti-human laminin α5 and anti-mouse IgG-Alexa Fluor 488, GBMs from transgenic mice (arrows), but not wildtype, were intensely immunolabeled. Transgenic and wildtype mice labeled with anti-mouse IgG alone as controls were negative (upper and lower right). Scale bar = 30μm.
    Figure Legend Snippet: Human laminin α5 is expressed in GBMs of hu LAMA5 transgenic mice. Representative sections of kidneys from littermates born from hu LAMA5 female mice crossed with wildtype males. When cryosections from hu LAMA5 transgenic pups (upper left) and wildtype mice (lower left) were immunolabeled with mouse anti-human laminin α5 and anti-mouse IgG-Alexa Fluor 488, GBMs from transgenic mice (arrows), but not wildtype, were intensely immunolabeled. Transgenic and wildtype mice labeled with anti-mouse IgG alone as controls were negative (upper and lower right). Scale bar = 30μm.

    Techniques Used: Transgenic Assay, Mouse Assay, Immunolabeling, Labeling

    Maternal sera contains anti-human kidney basement membrane IgG. Frozen sections of human kidney incubated with sera collected from two separate wildtype females mated two or more times with hu LAMA5 transgenic males (upper panels a and b), or with sera from a wildtype female before mating (c), or with sera from a hu LAMA5 transgenic female (d). All sections immunolabeled with anti-mouse IgG-Alexa Fluor 488. Sera from wildtype females X hu LAMA5 transgenic males binds human kidney basement membranes (a and b), but no binding is observed in sections incubated with sera collected before mating, or from hu LAMA5 transgenic females (c and d). Scale bar = 50μm.
    Figure Legend Snippet: Maternal sera contains anti-human kidney basement membrane IgG. Frozen sections of human kidney incubated with sera collected from two separate wildtype females mated two or more times with hu LAMA5 transgenic males (upper panels a and b), or with sera from a wildtype female before mating (c), or with sera from a hu LAMA5 transgenic female (d). All sections immunolabeled with anti-mouse IgG-Alexa Fluor 488. Sera from wildtype females X hu LAMA5 transgenic males binds human kidney basement membranes (a and b), but no binding is observed in sections incubated with sera collected before mating, or from hu LAMA5 transgenic females (c and d). Scale bar = 50μm.

    Techniques Used: Incubation, Transgenic Assay, Immunolabeling, Binding Assay

    34) Product Images from "The BNIP-2 and Cdc42GAP Homology (BCH) Domain of p50RhoGAP/Cdc42GAP Sequesters RhoA from Inactivation by the Adjacent GTPase-activating Protein Domain"

    Article Title: The BNIP-2 and Cdc42GAP Homology (BCH) Domain of p50RhoGAP/Cdc42GAP Sequesters RhoA from Inactivation by the Adjacent GTPase-activating Protein Domain

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E09-05-0408

    The BCH domain of p50RhoGAP inhibits GAP-induced cell rounding. (A) Schematic diagram of p50RhoGAP and its mutants: N-terminus without GAP domain (NBCH, amino acids 1-217), C-terminus without BCH domain (PGAP, amino acids 218-439), and a mutant without the proline-rich region, PPR (ΔPPR, Δ218-258). (B) HeLa cells were transfected with plasmids encoding FLAG-tagged full-length p50RhoGAP, NBCH, PGAP, or ΔPPR mutants. Cells were then fixed after 16–20 h and subjected to confocal fluorescence microscopy as described in Materials and Methods. Morphological changes and cytoskeletal rearrangements were revealed by indirect immunostaining with Alexa Fluor 488–conjugated goat anti-mouse IgG against anti-tubulin for microtubules and with FLAG antibody for expressed FLAG-tagged proteins. (C) For quantitative analysis, the ratio of cuboidal, protrusion/shrinkage, and round cells was scored with at least 150 transfected cells counted per sample per experiment. Data are means ± SD (n = 3).
    Figure Legend Snippet: The BCH domain of p50RhoGAP inhibits GAP-induced cell rounding. (A) Schematic diagram of p50RhoGAP and its mutants: N-terminus without GAP domain (NBCH, amino acids 1-217), C-terminus without BCH domain (PGAP, amino acids 218-439), and a mutant without the proline-rich region, PPR (ΔPPR, Δ218-258). (B) HeLa cells were transfected with plasmids encoding FLAG-tagged full-length p50RhoGAP, NBCH, PGAP, or ΔPPR mutants. Cells were then fixed after 16–20 h and subjected to confocal fluorescence microscopy as described in Materials and Methods. Morphological changes and cytoskeletal rearrangements were revealed by indirect immunostaining with Alexa Fluor 488–conjugated goat anti-mouse IgG against anti-tubulin for microtubules and with FLAG antibody for expressed FLAG-tagged proteins. (C) For quantitative analysis, the ratio of cuboidal, protrusion/shrinkage, and round cells was scored with at least 150 transfected cells counted per sample per experiment. Data are means ± SD (n = 3).

    Techniques Used: Mutagenesis, Transfection, Fluorescence, Microscopy, Immunostaining

    Three essential residues for GAP domain in inducing cell rounding. (A) HeLa cells were transfected with HA-tagged PGAP or the various mutants indicated, fixed after 20 h, and subjected to confocal fluorescence microscopy with anti-HA and Alexa Fluor 488 dye–conjugated goat anti-rabbit IgG as described in Materials and Methods . (B) To determine the Rho GTPase activity, HeLa cells were transfected with FLAG-tagged RhoA alone or with various HA-tagged PGAP or its various mutants. Cell were lysed and incubated with GST fusion of the Rho-binding domain of rhotekin immobilized on beads as described in Materials and Methods . Bound active RhoA were resolved on SDS-PAGE and detected by immunoblotting with FLAG-antibody (top panel). Equal loading of GST fusion proteins is shown in the second panel.
    Figure Legend Snippet: Three essential residues for GAP domain in inducing cell rounding. (A) HeLa cells were transfected with HA-tagged PGAP or the various mutants indicated, fixed after 20 h, and subjected to confocal fluorescence microscopy with anti-HA and Alexa Fluor 488 dye–conjugated goat anti-rabbit IgG as described in Materials and Methods . (B) To determine the Rho GTPase activity, HeLa cells were transfected with FLAG-tagged RhoA alone or with various HA-tagged PGAP or its various mutants. Cell were lysed and incubated with GST fusion of the Rho-binding domain of rhotekin immobilized on beads as described in Materials and Methods . Bound active RhoA were resolved on SDS-PAGE and detected by immunoblotting with FLAG-antibody (top panel). Equal loading of GST fusion proteins is shown in the second panel.

    Techniques Used: Transfection, Fluorescence, Microscopy, Activity Assay, Incubation, Binding Assay, SDS Page

    The BCH domain of p50RhoGAP inhibits the adjacent GAP function by sequestering RhoA. (A) HeLa cells were transfected for 20 h with FLAG-tagged p50RhoGAP wild type and mutants including FLBCH, FL121, FL161, FL181, or PGAP. Cells were then fixed and incubated with FLAG monoclonal antibodies, followed by Alexa Fluor 488-conjugated goat anti-mouse IgG. Cell morphology was monitored by direct staining with rhodamine-conjugated phalloidin for actin filaments. (B) For quantitative analysis, the ratio of cuboidal, protrusion/shrinkage, and round cells was scored and at least 150 transfected cells were counted per sample per experiment. Data are means ± SD (n = 3). (C) Cells were transfected with FLAG-tagged p50RhoGAP full-length, FL121, FL181, or PGAP in the presence of HA-RhoA. After 20 h, cell were lysed and incubated with GST fusion of the Rho-binding domain of rhotekin-immobilized on beads, in order to assess the impacts of p50RhoGAP and the mutants in regulating RhoA activity as described in Materials and Methods . Bound GTPases were resolved on SDS-PAGE and detected by immunoblotting with HA-antibody (top panel). Equal loading of GST fusion proteins is shown in the second panel. (D) HeLa cells were cotransfected with HA-tagged PGAP and FLAG-tagged-NBCH or BCH domain. Cells were then fixed and analyzed with confocal fluorescence microscopy as described in Materials and Methods .
    Figure Legend Snippet: The BCH domain of p50RhoGAP inhibits the adjacent GAP function by sequestering RhoA. (A) HeLa cells were transfected for 20 h with FLAG-tagged p50RhoGAP wild type and mutants including FLBCH, FL121, FL161, FL181, or PGAP. Cells were then fixed and incubated with FLAG monoclonal antibodies, followed by Alexa Fluor 488-conjugated goat anti-mouse IgG. Cell morphology was monitored by direct staining with rhodamine-conjugated phalloidin for actin filaments. (B) For quantitative analysis, the ratio of cuboidal, protrusion/shrinkage, and round cells was scored and at least 150 transfected cells were counted per sample per experiment. Data are means ± SD (n = 3). (C) Cells were transfected with FLAG-tagged p50RhoGAP full-length, FL121, FL181, or PGAP in the presence of HA-RhoA. After 20 h, cell were lysed and incubated with GST fusion of the Rho-binding domain of rhotekin-immobilized on beads, in order to assess the impacts of p50RhoGAP and the mutants in regulating RhoA activity as described in Materials and Methods . Bound GTPases were resolved on SDS-PAGE and detected by immunoblotting with HA-antibody (top panel). Equal loading of GST fusion proteins is shown in the second panel. (D) HeLa cells were cotransfected with HA-tagged PGAP and FLAG-tagged-NBCH or BCH domain. Cells were then fixed and analyzed with confocal fluorescence microscopy as described in Materials and Methods .

    Techniques Used: Transfection, Incubation, Staining, Binding Assay, Activity Assay, SDS Page, Fluorescence, Microscopy

    35) Product Images from "Antiviral activity of lambda-carrageenan against influenza viruses in mice and severe acute respiratory syndrome coronavirus 2 in vitro"

    Article Title: Antiviral activity of lambda-carrageenan against influenza viruses in mice and severe acute respiratory syndrome coronavirus 2 in vitro

    Journal: bioRxiv

    doi: 10.1101/2020.08.23.255364

    Anti-SARS-CoV-2 activity of λ-CGN. (A) Vero cells seeded in 96-well plates were infected with SARS-CoV-2 at an MOI of 0.1, either alone or in the presence of increasing concentrations of λ-CGN (upper panel) or RDV (lower panel; a control). On Day 2 post-infection, cells were fixed and permeabilized prior to immunostaining with an anti-viral S antibody and an Alexa Fluor 488-conjugated goat anti-mouse IgG (green). Cell nuclei were counterstained with DAPI to estimate cell viability (blue). Images were captured with a 20× objective lens fitted to an automated fluorescence microscope (Operetta HCS). (B) The number of fluorescent spots was counted to calculate antiviral activity (green) and cell viability (blue) at each concentration of the compounds. The viability of mock-infected cells were fixed as 100%, while the antiviral activity in virus-infected cells or mock-infected cells was fixed as 0 and 100%, respectively. Data are expressed as the mean ± SEM from independent experiments.
    Figure Legend Snippet: Anti-SARS-CoV-2 activity of λ-CGN. (A) Vero cells seeded in 96-well plates were infected with SARS-CoV-2 at an MOI of 0.1, either alone or in the presence of increasing concentrations of λ-CGN (upper panel) or RDV (lower panel; a control). On Day 2 post-infection, cells were fixed and permeabilized prior to immunostaining with an anti-viral S antibody and an Alexa Fluor 488-conjugated goat anti-mouse IgG (green). Cell nuclei were counterstained with DAPI to estimate cell viability (blue). Images were captured with a 20× objective lens fitted to an automated fluorescence microscope (Operetta HCS). (B) The number of fluorescent spots was counted to calculate antiviral activity (green) and cell viability (blue) at each concentration of the compounds. The viability of mock-infected cells were fixed as 100%, while the antiviral activity in virus-infected cells or mock-infected cells was fixed as 0 and 100%, respectively. Data are expressed as the mean ± SEM from independent experiments.

    Techniques Used: Activity Assay, Infection, Immunostaining, Fluorescence, Microscopy, Concentration Assay

    Effect of λ-CGN on the intracellular entry of influenza A virus. MDCK cells were infected with PR8 (MOI, 5) and subsequently mock-treated or treated either with λ-CGN or with p-KG03 at a concentration of 10 μg/ml. At 4 h p.i. in the absence of CHX (A) or at 2.5 h in the presence of 10 μg/ml CHX (B), viral NP was detected with an anti-NP antibody and an Alex Fluor 488-conjugated goat anti-mouse secondary antibody (green). Cell nuclei were counterstained with DAPI (blue). Original magnification, 400×.
    Figure Legend Snippet: Effect of λ-CGN on the intracellular entry of influenza A virus. MDCK cells were infected with PR8 (MOI, 5) and subsequently mock-treated or treated either with λ-CGN or with p-KG03 at a concentration of 10 μg/ml. At 4 h p.i. in the absence of CHX (A) or at 2.5 h in the presence of 10 μg/ml CHX (B), viral NP was detected with an anti-NP antibody and an Alex Fluor 488-conjugated goat anti-mouse secondary antibody (green). Cell nuclei were counterstained with DAPI (blue). Original magnification, 400×.

    Techniques Used: Infection, Concentration Assay

    36) Product Images from "Nanoassembly routes stimulate conflicting antibody quantity and quality for transmission-blocking malaria vaccines"

    Article Title: Nanoassembly routes stimulate conflicting antibody quantity and quality for transmission-blocking malaria vaccines

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-03798-3

    Binding of anti-Pfs25 IgG to native antigen. Ookinetes of P . berghei transgenic for Pfs25 (Pfs25DR3) were stained with 4B7 (positive control) or pooled post boost sera from mice immunised with one of the nanoparticle vaccines, monomeric Pfs25 or ovalbumin (OVA) (negative control). Antibody binding was detected by Alexa Fluor® 488-conjugated goat anti-mouse IgG (green) and the DNA was stained with DAPI (blue). Phase contrast as well as merged view are also shown. White bars correspond to 5 μm.
    Figure Legend Snippet: Binding of anti-Pfs25 IgG to native antigen. Ookinetes of P . berghei transgenic for Pfs25 (Pfs25DR3) were stained with 4B7 (positive control) or pooled post boost sera from mice immunised with one of the nanoparticle vaccines, monomeric Pfs25 or ovalbumin (OVA) (negative control). Antibody binding was detected by Alexa Fluor® 488-conjugated goat anti-mouse IgG (green) and the DNA was stained with DAPI (blue). Phase contrast as well as merged view are also shown. White bars correspond to 5 μm.

    Techniques Used: Binding Assay, Transgenic Assay, Staining, Positive Control, Mouse Assay, Negative Control

    37) Product Images from "Establishment of an immunoscreening system using recombinant VP1 protein for the isolation of a monoclonal antibody that blocks JC virus infection"

    Article Title: Establishment of an immunoscreening system using recombinant VP1 protein for the isolation of a monoclonal antibody that blocks JC virus infection

    Journal: Biochemical and Biophysical Research Communications

    doi: 10.1016/j.bbrc.2004.11.158

    Characterization of the molecule recognized by 24D2. (A) Immunoblotting analysis with membrane extracts derived from IMR-32 cells. Aliquots of 20 μg of the detergent-solubilized IMR-32 membrane fraction were loaded and immunoblotted with 24D2 at a dilution of 1:60. (B) Immunoblotting analysis with various cell lysates. The membrane fractions were prepared from IMR-32, HEK293, COS7, HeLa, and SVG-A cells. Molecular size markers are indicated on the left of the column. (C) Subcellular localization of the molecule recognized by 24D2 in IMR-32 and HEK-293 cells. Methanol-fixed cells were incubated with 24D2 at a dilution of 1:60, following Alexa Fluor 488-conjugated goat anti-mouse IgM. The immunofluorescence signal is represented as a green color. The lower panels show higher magnifications of the upper panels. Bars, 20 μm (upper panels) and 10 μm (lower panels).
    Figure Legend Snippet: Characterization of the molecule recognized by 24D2. (A) Immunoblotting analysis with membrane extracts derived from IMR-32 cells. Aliquots of 20 μg of the detergent-solubilized IMR-32 membrane fraction were loaded and immunoblotted with 24D2 at a dilution of 1:60. (B) Immunoblotting analysis with various cell lysates. The membrane fractions were prepared from IMR-32, HEK293, COS7, HeLa, and SVG-A cells. Molecular size markers are indicated on the left of the column. (C) Subcellular localization of the molecule recognized by 24D2 in IMR-32 and HEK-293 cells. Methanol-fixed cells were incubated with 24D2 at a dilution of 1:60, following Alexa Fluor 488-conjugated goat anti-mouse IgM. The immunofluorescence signal is represented as a green color. The lower panels show higher magnifications of the upper panels. Bars, 20 μm (upper panels) and 10 μm (lower panels).

    Techniques Used: Derivative Assay, Incubation, Immunofluorescence

    38) Product Images from "Stat1 Phosphorylation Determines Ras Oncogenicity by Regulating p27Kip1"

    Article Title: Stat1 Phosphorylation Determines Ras Oncogenicity by Regulating p27Kip1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0003476

    p27 Kip1 contributes to the inhibition of cell cycle progression by Stat1. (A) MEFs maintained at 70% confluence were subjected to immunostaining with an anti-p27 Kip1 mAb and a goat anti-mouse IgG conjugated to Alexa Fluro 488 (green). The nucleus was visualized by 4,6-diamidino-2-phenylindole (DAPI) staining. (B) Protein extracts (500 µg) from MEFs that reached 50% (lanes 1–4) or 90% confluence (lanes 5–8) were subjected to immunoprecipitation with an anti-Cyclin E antibody followed by in vitro kinase assays using GST-Rb (1 µg) and 1 µCi of [γ- 32 P] ATP (panel a). GST-Rb protein levels were visualized by Commassie blue staining (panel b). The levels of Cyclin E (panel c) and p27 Kip1 (panel d) in the kinase assays were detected by immunoblotting. CyclinE-Cdk2 activity was assessed by normalizing GST-Rb phosphorylation levels to GST-Rb protein levels. The graph shows results expressed as ±SD from 3 independent experiments (*P
    Figure Legend Snippet: p27 Kip1 contributes to the inhibition of cell cycle progression by Stat1. (A) MEFs maintained at 70% confluence were subjected to immunostaining with an anti-p27 Kip1 mAb and a goat anti-mouse IgG conjugated to Alexa Fluro 488 (green). The nucleus was visualized by 4,6-diamidino-2-phenylindole (DAPI) staining. (B) Protein extracts (500 µg) from MEFs that reached 50% (lanes 1–4) or 90% confluence (lanes 5–8) were subjected to immunoprecipitation with an anti-Cyclin E antibody followed by in vitro kinase assays using GST-Rb (1 µg) and 1 µCi of [γ- 32 P] ATP (panel a). GST-Rb protein levels were visualized by Commassie blue staining (panel b). The levels of Cyclin E (panel c) and p27 Kip1 (panel d) in the kinase assays were detected by immunoblotting. CyclinE-Cdk2 activity was assessed by normalizing GST-Rb phosphorylation levels to GST-Rb protein levels. The graph shows results expressed as ±SD from 3 independent experiments (*P

    Techniques Used: Inhibition, Immunostaining, Staining, Immunoprecipitation, In Vitro, Activity Assay

    39) Product Images from "Membrane skeleton orchestrates the platelet glycoprotein (GP) Ib‐IX complex clustering and signaling"

    Article Title: Membrane skeleton orchestrates the platelet glycoprotein (GP) Ib‐IX complex clustering and signaling

    Journal: Iubmb Life

    doi: 10.1002/iub.1559

    The membrane skeleton restricts the clustering of the GP Ib‐IX complex upon antibody crosslinking. (A) Wild‐type GP Ibα WT , ABP‐280‐binding deficient GP Ibα FW‐AA (Phe568Ala and Trp570Ala) and CT‐truncated GP Ibα ΔPhe555 were retro‐virally transfected into β WT IX WT ‐expressing K562 cells. Flow cytometry analysis with a phycoerythrin‐labeled anti‐GP Ibα antibody showed that GP Ibα can be efficiently expressed with comparable levels in the βIX‐harboring K562 cells. (B) Cells were first incubated with the mouse anti‐GP Ibα monoclonal antibody (SZ2) or an isotype‐matched mouse antibody, and then left untreated (a, c, e, and g) or treated (b, d, f, and h) by crosslinking with an Alexa Fluor 488–labeled goat anti‐mouse secondary antibody. In the absence of antibody crosslinking, the GP Ibα evenly distributes on all GP Ibα‐expressing cells (c, e, and g) with little sign of GP Ibα clusters in any of these cells, demonstrating that loss of the membrane skeletal constraint does not cause a passive clustering of the surface GP Ibα. In comparison, antibody crosslinking causes the formation of positive GP Ibα‐staining clusters where minor difference in the size or distribution patterns of these clusters were shown between wild‐type GP Ibα WT and ABP‐280‐binding deficient GP Ibα FW‐AA expressing cells (d and f). In sharp contrast, large punctuated GP Ibα‐positive clusters appeared in the CT‐truncated GP Ibα ΔPhe555 cells upon antibody crosslinking (h), suggesting that additional force(s) other than the ABP‐280‐mediated membrane skeletal constraint may exist to restrict the movement of the GP Ibα on the cell surface.
    Figure Legend Snippet: The membrane skeleton restricts the clustering of the GP Ib‐IX complex upon antibody crosslinking. (A) Wild‐type GP Ibα WT , ABP‐280‐binding deficient GP Ibα FW‐AA (Phe568Ala and Trp570Ala) and CT‐truncated GP Ibα ΔPhe555 were retro‐virally transfected into β WT IX WT ‐expressing K562 cells. Flow cytometry analysis with a phycoerythrin‐labeled anti‐GP Ibα antibody showed that GP Ibα can be efficiently expressed with comparable levels in the βIX‐harboring K562 cells. (B) Cells were first incubated with the mouse anti‐GP Ibα monoclonal antibody (SZ2) or an isotype‐matched mouse antibody, and then left untreated (a, c, e, and g) or treated (b, d, f, and h) by crosslinking with an Alexa Fluor 488–labeled goat anti‐mouse secondary antibody. In the absence of antibody crosslinking, the GP Ibα evenly distributes on all GP Ibα‐expressing cells (c, e, and g) with little sign of GP Ibα clusters in any of these cells, demonstrating that loss of the membrane skeletal constraint does not cause a passive clustering of the surface GP Ibα. In comparison, antibody crosslinking causes the formation of positive GP Ibα‐staining clusters where minor difference in the size or distribution patterns of these clusters were shown between wild‐type GP Ibα WT and ABP‐280‐binding deficient GP Ibα FW‐AA expressing cells (d and f). In sharp contrast, large punctuated GP Ibα‐positive clusters appeared in the CT‐truncated GP Ibα ΔPhe555 cells upon antibody crosslinking (h), suggesting that additional force(s) other than the ABP‐280‐mediated membrane skeletal constraint may exist to restrict the movement of the GP Ibα on the cell surface.

    Techniques Used: Binding Assay, Transfection, Expressing, Flow Cytometry, Cytometry, Labeling, Incubation, Staining

    The membrane GEM domain is not essential for antibody‐crosslinking induced GP Ibα clustering. Wild‐type GP Ibα WT and CT‐truncated GP Ibα ΔPhe555 were untreated or pretreated with MβCD, a known cholesterol depriving and GEMs disrupting reagent, followed by SZ2 staining and antibody crosslinking, as described in Fig. 1. After fixation, the membrane distribution of the GEM domain was revealed by counterstaining the cells with Alexa Fluor® 488 conjugated CT‐B. In both cells the GP Ibα clusters (green, b, e, h, and k) co‐localized well with the CT‐B patches (red, c, f, i, and l), where all GP Ibα clusters associated with the CT‐B patches (merged, a, d, g, and j). Larger punctuated GEMs patches were formed in the GP Ibα ΔPhe555 cells than those in GP Ibα WT cells (c and i), suggesting the GEMs forms patches when GP Ibα clusters. Treatment with MβCD did not affect GP Ibα clustering (e and k) but abolished the GEMs (f and l) and the GP Ibα/GEMs‐colocalizing structures in both cells (merged, d and j), indicating that the clustering of GP Ibα does not depend on the GP Ibα‐associating GEMs, instead, on the presence of the membrane skeletal constraint.
    Figure Legend Snippet: The membrane GEM domain is not essential for antibody‐crosslinking induced GP Ibα clustering. Wild‐type GP Ibα WT and CT‐truncated GP Ibα ΔPhe555 were untreated or pretreated with MβCD, a known cholesterol depriving and GEMs disrupting reagent, followed by SZ2 staining and antibody crosslinking, as described in Fig. 1. After fixation, the membrane distribution of the GEM domain was revealed by counterstaining the cells with Alexa Fluor® 488 conjugated CT‐B. In both cells the GP Ibα clusters (green, b, e, h, and k) co‐localized well with the CT‐B patches (red, c, f, i, and l), where all GP Ibα clusters associated with the CT‐B patches (merged, a, d, g, and j). Larger punctuated GEMs patches were formed in the GP Ibα ΔPhe555 cells than those in GP Ibα WT cells (c and i), suggesting the GEMs forms patches when GP Ibα clusters. Treatment with MβCD did not affect GP Ibα clustering (e and k) but abolished the GEMs (f and l) and the GP Ibα/GEMs‐colocalizing structures in both cells (merged, d and j), indicating that the clustering of GP Ibα does not depend on the GP Ibα‐associating GEMs, instead, on the presence of the membrane skeletal constraint.

    Techniques Used: Staining

    40) Product Images from "Inhibition of Melanogenesis by the Pyridinyl Imidazole Class of Compounds: Possible Involvement of the Wnt/?-Catenin Signaling Pathway"

    Article Title: Inhibition of Melanogenesis by the Pyridinyl Imidazole Class of Compounds: Possible Involvement of the Wnt/?-Catenin Signaling Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0033021

    Regulation of Wnt/β-catenin signaling by pyridinyl imidazoles. (A) Inhibition of the β-catenin/Tcf/Lef1-responsive luciferase reporter gene by PI compounds. The pTK-Renilla was inserted as an internal control. Twenty-four hours after transfection, cells were treated with PI compounds (SB202474, SB202190, SB203580, SB220025, PD169316 20 µM: MAPK Inh III 10 µM) for 6 h. Firefly luciferase activity, normalized to the corresponding renilla luciferase activity was expressed as fold decrease compared with control cells. Values represent mean ± SD of three representative experiments performed in duplicate. (B) Semi-qunatitative real-time PCR was used to measure Wnt/β-catenin-target genes Axin2, Lef1 and Wisp1 mRNAs expression in B16-F0 cells after 6 h of treatments with PI compounds. The graphs show fold differences in transcript abundance in comparison with untreated cells. Results shown were normalized by the β-actin mRNA levels. The data show the mean±SD of three experiments performed in triplicate. *P≤0.05; #P≤0.01 versus control. (C) Expression of β-catenin in B16-F0 cells after 6 h of treatment with PI compounds (SB202474, SB202190, SB203580, SB220025, PD169316 20 µM: MAPK Inh III 10 µM). Total cellular proteins (30 µg/lane) were subject to 10% SDS-PAGE. Variation of loading was determined by blotting with anti-β-tubulin antibody. Western blot assays are representative of at least three experiments. (D) Immunofluorescence analysis of β-catenin. B16-F0 cells were grown on glass coverslips and then treated with SB202474, PD169316 (20 µM) or DMSO respectively. Six hours later, cells were fixed and analyzed by immunofluorescence labelling with a mouse monoclonal anti-β-catenin followed by Alexa-Fluor-546-conjugated goat anti-mouse IgG antibody. Nuclei were labelled with bisbenzidine (DAPI). Original magnification 20×.
    Figure Legend Snippet: Regulation of Wnt/β-catenin signaling by pyridinyl imidazoles. (A) Inhibition of the β-catenin/Tcf/Lef1-responsive luciferase reporter gene by PI compounds. The pTK-Renilla was inserted as an internal control. Twenty-four hours after transfection, cells were treated with PI compounds (SB202474, SB202190, SB203580, SB220025, PD169316 20 µM: MAPK Inh III 10 µM) for 6 h. Firefly luciferase activity, normalized to the corresponding renilla luciferase activity was expressed as fold decrease compared with control cells. Values represent mean ± SD of three representative experiments performed in duplicate. (B) Semi-qunatitative real-time PCR was used to measure Wnt/β-catenin-target genes Axin2, Lef1 and Wisp1 mRNAs expression in B16-F0 cells after 6 h of treatments with PI compounds. The graphs show fold differences in transcript abundance in comparison with untreated cells. Results shown were normalized by the β-actin mRNA levels. The data show the mean±SD of three experiments performed in triplicate. *P≤0.05; #P≤0.01 versus control. (C) Expression of β-catenin in B16-F0 cells after 6 h of treatment with PI compounds (SB202474, SB202190, SB203580, SB220025, PD169316 20 µM: MAPK Inh III 10 µM). Total cellular proteins (30 µg/lane) were subject to 10% SDS-PAGE. Variation of loading was determined by blotting with anti-β-tubulin antibody. Western blot assays are representative of at least three experiments. (D) Immunofluorescence analysis of β-catenin. B16-F0 cells were grown on glass coverslips and then treated with SB202474, PD169316 (20 µM) or DMSO respectively. Six hours later, cells were fixed and analyzed by immunofluorescence labelling with a mouse monoclonal anti-β-catenin followed by Alexa-Fluor-546-conjugated goat anti-mouse IgG antibody. Nuclei were labelled with bisbenzidine (DAPI). Original magnification 20×.

    Techniques Used: Inhibition, Luciferase, Transfection, Activity Assay, Real-time Polymerase Chain Reaction, Expressing, SDS Page, Western Blot, Immunofluorescence

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    Incubation:

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    Article Snippet: For fluorescent staining, the smears were incubated in 0.1% triton X-100 solution for 5 min. To inhibit non-specific staining, the smears were incubated with 10% (v/v) normal horse serum for 30 min. To demonstrate the presence of luteal cells in lymphatic fluid, the smear was incubated with 3β-HSD antibody diluted at 1∶500 with PBS for 30 min at RT. .. Then the smear was incubated with Alexa Fluor 488 Goat anti-mouse IgG (A-11001; Life Technologies, Carlsbad, CA, USA) diluted at 1∶1000 with PBS for 30 min at RT. .. The smear was incubated with Bodipy 493/503 (D-3922; Life Technologies, Carlsbad, CA, USA) used for lipid droplets-staining diluted at 1∶200 with PBS for 20 min at RT.

    Article Title: Expression of CD133 in SW620 colorectal cancer cells is modulated by the microenvironment
    Article Snippet: The resulting spheroids were photographed in phase contrast using a Nikon microscope with a ×10 lens. .. The sorted cells were seeded on glass cover slips, fixed in 4% paraformaldehyde and blocked in PBS with 0.2% BSA for 30 min at room temperature prior to incubation with mouse anti-human CD133/1 antibody (1:40; Miltenyi Biotec) followed by goat anti-mouse Alexa488 (Invitrogen, Carlsbad, CA, USA). .. The nuclei of the cells were stained by incubating with DAPI (1 μg/ml) for 5 min.

    Article Title: Prostacyclin Prevents Pericyte Loss and Demyelination Induced by Lysophosphatidylcholine in the Central Nervous System *
    Article Snippet: .. Sections were incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG (1:500, Invitrogen, catalog no. A-11001) and DyLight 594-labeled L. esculentum (tomato) lectin (1:100) for 1 h at room temperature. .. Mice were intravenously injected with 0.1 ml of Alexa Fluor 555-conjugated cadaverine at a concentration of 0.5 mg/ml (Life Technologies, Inc.) under anesthesia.

    Article Title: Evidence of the presence of nucleic acids and β-glucan in the matrix of non-typeable Haemophilus influenzae in vitro biofilms
    Article Snippet: The biofilms were stained with ConA-Alexa fluor 647 (C21421, Invitrogen), HPA-Alexa fluor 488 (L11271, Invitrogen), PNA-Alexa fluor 594 (L32459, Invitrogen), SBA-Alexa fluor 488 (L11272, Invitrogen) or WGA-Alexa fluor 488 (W11261, Invitrogen) at 5–25 μg mL–1 each. .. All staining procedures involved incubation for 10–20 min at room temperature in the dark, except when biofilms were incubated with mouse anti-dsDNA antibody (2 μg mL–1 ); this involved 1 h incubation at 4 °C followed by 30 min incubation at room temperature in the dark with Alexa fluor 488-labelled goat anti-mouse IgG (1:500) (A-11001, Invitrogen) (diluted 1/500). .. Lectins and CW were incubated for 20 min with 25–80 mg mL−1 of specific sugars or polysaccharides at room temperature in the dark to check their binding .

    Labeling:

    Article Title: Salinomycin Inhibits Influenza Virus Infection by Disrupting Endosomal Acidification and Viral Matrix Protein 2 Function
    Article Snippet: After blocking with 1% bovine serum albumin (BSA) and 10% normal goat serum prepared in PBS, the slides were incubated overnight at 4°C with an influenza A virus NP-specific monoclonal antibody (catalog no. sc-80481; Santa Cruz Biotechnology). .. The anti-NP antibody-bound cells were subsequently labeled for 1 h at room temperature with Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen) and counterstained with Vectashield mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA). .. Laser scanning confocal microscopy was performed with a Zeiss LSM 700 confocal microscope.

    Staining:

    Article Title: Evidence of the presence of nucleic acids and β-glucan in the matrix of non-typeable Haemophilus influenzae in vitro biofilms
    Article Snippet: The biofilms were stained with ConA-Alexa fluor 647 (C21421, Invitrogen), HPA-Alexa fluor 488 (L11271, Invitrogen), PNA-Alexa fluor 594 (L32459, Invitrogen), SBA-Alexa fluor 488 (L11272, Invitrogen) or WGA-Alexa fluor 488 (W11261, Invitrogen) at 5–25 μg mL–1 each. .. All staining procedures involved incubation for 10–20 min at room temperature in the dark, except when biofilms were incubated with mouse anti-dsDNA antibody (2 μg mL–1 ); this involved 1 h incubation at 4 °C followed by 30 min incubation at room temperature in the dark with Alexa fluor 488-labelled goat anti-mouse IgG (1:500) (A-11001, Invitrogen) (diluted 1/500). .. Lectins and CW were incubated for 20 min with 25–80 mg mL−1 of specific sugars or polysaccharides at room temperature in the dark to check their binding .

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    Thermo Fisher alexa fluor 488 conjugated goat anti mouse igg antibody
    Chemical structures of the compounds tested and visualization of SARS-CoV-2-infected Vero cells. ( A ) Chemical structures of gemcitabine, 2FdC, and remdesivir. ( B ) MTT-based cytotoxicity assay of gemcitabine at different concentrations and time points. Vero cells were treated with increasing concentrations of gemcitabine for 24 h (black square) or 48 h (gray square). Percentage cell viability was measured by using MTT, in which mock-treated cells served as a control (100%). ( C ) Fluorescein diacetate-based cytotoxicity assay of gemcitabine. Vero cells were treated with increasing concentrations of gemcitabine for 24 (black square) and 48 h (gray square). Percentage cell viability was measured by addition of fluorescein diacetate, in which mock-treated cells served as a control (100%). ( D ) Fluorescein diacetate-based cytotoxicity assay of a delivery vehicle. Increasing concentrations of DMSO, 0.2, 0.6 and 1.8% ( v / v ), that were identically included in 100, 300 and 900 μM gemcitabine shown in ( C ), were treated to Vero cells for 24 (black bar) and 48 h (gray bar). Values in ( B – D ) are means ± standard deviations from thee three independent experiments. ( E ) Visualization of SARS-CoV-2 infection. Vero cells were mock-infected (Mock) or infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.02 for 24 h. Viral spike (S) protein was probed with mouse anti-S antibody and <t>Alexa</t> Fluor 488-conjugated goat anti-mouse antibody (green). Cellular nuclei were counterstained with DAPI (blue). Magnification ×20.
    Alexa Fluor 488 Conjugated Goat Anti Mouse Igg Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexa fluor 488 conjugated goat anti mouse igg
    Treatment with iloprost prevents LPC-mediated vascular barrier disruption in the adult spinal cord. A , visualization of vascular leakage in the CNS. Upper panels, representative images of the thoracic spinal cord showing leakage of <t>Alexa</t> Fluor 555-conjugated cadaverine ( red ) into the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. The broken line represents the outline of the tissue. Scale bar, 1 mm. Middle panels, representative images of the thoracic spinal cord showing the distribution of Alexa Fluor 555-conjugated cadaverine ( red ) in the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. Sections were counterstained for CD31 (vascular endothelial cell marker, green ). Scale bar, 100 μm. Lower panels, representative images of cross-sections of the thoracic spinal cord showing leakage of endogenous <t>IgG</t> ( green ) into the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. Sections were vascular counterstained with DyLight 594-labeled L. esculentum lectin (vascular endothelial cell marker, red ). Scale bar, 100 μm. B , quantification of Evans Blue leakage in lesions of the spinal cord at 1 day after the operation. Values represent the mean ± S.E. of three independent experiments. **, p
    Alexa Fluor 488 Conjugated Goat Anti Mouse Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexa fluor 488 conjugated goat anti mouse igg secondary antibody
    Binding of DENV NS1 to Mouse Tissues Cryo-sections of mouse (A) lung, (B) liver, and (C) intestine were incubated with serum-free supernatants from BHK DENV-2 Rep or BHK cells for 1 h at room temperature. After extensive washing, bound NS1 was detected by a mixture of NS1 mAbs (1A4, 1F11, 2G6, 1B2) followed by Cy3-conjugated goat anti-mouse <t>IgG.</t> Co-staining with endothelial cell marker was subsequently performed by incubating the sections with rat anti-mouse CD31 (PECAM-1) followed by <t>Alexa</t> Fluor 488-conjugated goat anti-rat IgG. Nuclei were stained with a DNA-specific dye TO-PRO-3. Sections incubated with DENV NS1 followed by an isotype control Ab served as a negative control. Analysis was performed by confocal microscopy. White arrow and yellow arrowhead denote the layer of endothelial cells in the lumen and the outer layer of the adventitia of pulmonary vessel, respectively.
    Alexa Fluor 488 Conjugated Goat Anti Mouse Igg Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 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
    Direct injection results in elevated S1P levels which correlate with the activation of receptor 1 in muscle fibers. ( A ) To quantify the elevation of S1P following direct administration, we injected a single dose (same dose as Figure 5 ) of S1P in left TAs and vehicle in right TAs of uninjured mdx 4cv (n = 3, 11-MO) mice. TA muscles were harvested 15 minutes post injection for analysis by LC-MS/MS. Results indicate a significant elevation of S1P following direct injection. ( B ) To visualize the location of S1P following injection, biotinylated-S1P was injected in left TAs versus vehicle in right TAs of uninjured mdx 4cv mice (n = 2, 11-MO). Once more, TAs were harvested 15 minutes following injection. Staining with streptavidin conjugated to <t>Alexa</t> Fluor 594 reveals the presence of S1P-biotin around the perimeter of muscle fibers. ( C ) Staining of mdx 4cv TAs for S1PR1 and S1PR3 reveals S1PR1 is localized to the perimeter and perinuclear area (arrow) of muscle fibers (left photo). In contrast, staining for S1PR3 was mainly localized to the muscle vasculature (middle photo). Staining in parallel with an <t>IgG</t> isotype control for both antibodies shows the absence of non-specific staining (right graph). ( D ) Staining for S1PR1 in CTX-injured TAs (same tissue from Figure 5 ) reveals S1PR1 is present at the perimeter and perinuclear area of regenerating eMyHC+ fibers. ( E ) Staining for phosphorylated S1PR1 in the same mdx 4cv TAs was more prominent in the perinuclear area of eMyHC+ fibers, indicating the presence of active S1PR1 signaling in regenerating fibers. Scale bars = 50 μm. ** P
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    Image Search Results


    Chemical structures of the compounds tested and visualization of SARS-CoV-2-infected Vero cells. ( A ) Chemical structures of gemcitabine, 2FdC, and remdesivir. ( B ) MTT-based cytotoxicity assay of gemcitabine at different concentrations and time points. Vero cells were treated with increasing concentrations of gemcitabine for 24 h (black square) or 48 h (gray square). Percentage cell viability was measured by using MTT, in which mock-treated cells served as a control (100%). ( C ) Fluorescein diacetate-based cytotoxicity assay of gemcitabine. Vero cells were treated with increasing concentrations of gemcitabine for 24 (black square) and 48 h (gray square). Percentage cell viability was measured by addition of fluorescein diacetate, in which mock-treated cells served as a control (100%). ( D ) Fluorescein diacetate-based cytotoxicity assay of a delivery vehicle. Increasing concentrations of DMSO, 0.2, 0.6 and 1.8% ( v / v ), that were identically included in 100, 300 and 900 μM gemcitabine shown in ( C ), were treated to Vero cells for 24 (black bar) and 48 h (gray bar). Values in ( B – D ) are means ± standard deviations from thee three independent experiments. ( E ) Visualization of SARS-CoV-2 infection. Vero cells were mock-infected (Mock) or infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.02 for 24 h. Viral spike (S) protein was probed with mouse anti-S antibody and Alexa Fluor 488-conjugated goat anti-mouse antibody (green). Cellular nuclei were counterstained with DAPI (blue). Magnification ×20.

    Journal: International Journal of Molecular Sciences

    Article Title: Comparison of Antiviral Activity of Gemcitabine with 2′-Fluoro-2′-Deoxycytidine and Combination Therapy with Remdesivir against SARS-CoV-2

    doi: 10.3390/ijms22041581

    Figure Lengend Snippet: Chemical structures of the compounds tested and visualization of SARS-CoV-2-infected Vero cells. ( A ) Chemical structures of gemcitabine, 2FdC, and remdesivir. ( B ) MTT-based cytotoxicity assay of gemcitabine at different concentrations and time points. Vero cells were treated with increasing concentrations of gemcitabine for 24 h (black square) or 48 h (gray square). Percentage cell viability was measured by using MTT, in which mock-treated cells served as a control (100%). ( C ) Fluorescein diacetate-based cytotoxicity assay of gemcitabine. Vero cells were treated with increasing concentrations of gemcitabine for 24 (black square) and 48 h (gray square). Percentage cell viability was measured by addition of fluorescein diacetate, in which mock-treated cells served as a control (100%). ( D ) Fluorescein diacetate-based cytotoxicity assay of a delivery vehicle. Increasing concentrations of DMSO, 0.2, 0.6 and 1.8% ( v / v ), that were identically included in 100, 300 and 900 μM gemcitabine shown in ( C ), were treated to Vero cells for 24 (black bar) and 48 h (gray bar). Values in ( B – D ) are means ± standard deviations from thee three independent experiments. ( E ) Visualization of SARS-CoV-2 infection. Vero cells were mock-infected (Mock) or infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.02 for 24 h. Viral spike (S) protein was probed with mouse anti-S antibody and Alexa Fluor 488-conjugated goat anti-mouse antibody (green). Cellular nuclei were counterstained with DAPI (blue). Magnification ×20.

    Article Snippet: Viral S protein was probed using anti-S antibody (Cat. No., GTX632604; Genetex, Irvine, CA, USA) and Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Invitrogen, Carlsbad, CA, USA), while cellular nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen).

    Techniques: Infection, MTT Assay, Cytotoxicity Assay

    Treatment with iloprost prevents LPC-mediated vascular barrier disruption in the adult spinal cord. A , visualization of vascular leakage in the CNS. Upper panels, representative images of the thoracic spinal cord showing leakage of Alexa Fluor 555-conjugated cadaverine ( red ) into the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. The broken line represents the outline of the tissue. Scale bar, 1 mm. Middle panels, representative images of the thoracic spinal cord showing the distribution of Alexa Fluor 555-conjugated cadaverine ( red ) in the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. Sections were counterstained for CD31 (vascular endothelial cell marker, green ). Scale bar, 100 μm. Lower panels, representative images of cross-sections of the thoracic spinal cord showing leakage of endogenous IgG ( green ) into the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. Sections were vascular counterstained with DyLight 594-labeled L. esculentum lectin (vascular endothelial cell marker, red ). Scale bar, 100 μm. B , quantification of Evans Blue leakage in lesions of the spinal cord at 1 day after the operation. Values represent the mean ± S.E. of three independent experiments. **, p

    Journal: The Journal of Biological Chemistry

    Article Title: Prostacyclin Prevents Pericyte Loss and Demyelination Induced by Lysophosphatidylcholine in the Central Nervous System *

    doi: 10.1074/jbc.M114.587253

    Figure Lengend Snippet: Treatment with iloprost prevents LPC-mediated vascular barrier disruption in the adult spinal cord. A , visualization of vascular leakage in the CNS. Upper panels, representative images of the thoracic spinal cord showing leakage of Alexa Fluor 555-conjugated cadaverine ( red ) into the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. The broken line represents the outline of the tissue. Scale bar, 1 mm. Middle panels, representative images of the thoracic spinal cord showing the distribution of Alexa Fluor 555-conjugated cadaverine ( red ) in the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. Sections were counterstained for CD31 (vascular endothelial cell marker, green ). Scale bar, 100 μm. Lower panels, representative images of cross-sections of the thoracic spinal cord showing leakage of endogenous IgG ( green ) into the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. Sections were vascular counterstained with DyLight 594-labeled L. esculentum lectin (vascular endothelial cell marker, red ). Scale bar, 100 μm. B , quantification of Evans Blue leakage in lesions of the spinal cord at 1 day after the operation. Values represent the mean ± S.E. of three independent experiments. **, p

    Article Snippet: Sections were incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG (1:500, Invitrogen, catalog no. A-11001) and DyLight 594-labeled L. esculentum (tomato) lectin (1:100) for 1 h at room temperature.

    Techniques: Injection, Marker, Labeling

    Binding of DENV NS1 to Mouse Tissues Cryo-sections of mouse (A) lung, (B) liver, and (C) intestine were incubated with serum-free supernatants from BHK DENV-2 Rep or BHK cells for 1 h at room temperature. After extensive washing, bound NS1 was detected by a mixture of NS1 mAbs (1A4, 1F11, 2G6, 1B2) followed by Cy3-conjugated goat anti-mouse IgG. Co-staining with endothelial cell marker was subsequently performed by incubating the sections with rat anti-mouse CD31 (PECAM-1) followed by Alexa Fluor 488-conjugated goat anti-rat IgG. Nuclei were stained with a DNA-specific dye TO-PRO-3. Sections incubated with DENV NS1 followed by an isotype control Ab served as a negative control. Analysis was performed by confocal microscopy. White arrow and yellow arrowhead denote the layer of endothelial cells in the lumen and the outer layer of the adventitia of pulmonary vessel, respectively.

    Journal: PLoS Pathogens

    Article Title: Secreted NS1 of Dengue Virus Attaches to the Surface of Cells via Interactions with Heparan Sulfate and Chondroitin Sulfate E

    doi: 10.1371/journal.ppat.0030183

    Figure Lengend Snippet: Binding of DENV NS1 to Mouse Tissues Cryo-sections of mouse (A) lung, (B) liver, and (C) intestine were incubated with serum-free supernatants from BHK DENV-2 Rep or BHK cells for 1 h at room temperature. After extensive washing, bound NS1 was detected by a mixture of NS1 mAbs (1A4, 1F11, 2G6, 1B2) followed by Cy3-conjugated goat anti-mouse IgG. Co-staining with endothelial cell marker was subsequently performed by incubating the sections with rat anti-mouse CD31 (PECAM-1) followed by Alexa Fluor 488-conjugated goat anti-rat IgG. Nuclei were stained with a DNA-specific dye TO-PRO-3. Sections incubated with DENV NS1 followed by an isotype control Ab served as a negative control. Analysis was performed by confocal microscopy. White arrow and yellow arrowhead denote the layer of endothelial cells in the lumen and the outer layer of the adventitia of pulmonary vessel, respectively.

    Article Snippet: After three washes, sections were incubated with Alexa Fluor 488 conjugated goat anti-mouse IgG secondary antibody (Invitrogen) and followed by 1-h incubation at room temperature with rabbit anti-human CD31 (PECAM) (Santa Cruz Biotechnology) at the dilution of 1:10.

    Techniques: Binding Assay, Incubation, Staining, Marker, Negative Control, Confocal Microscopy

    Binding of DENV NS1 to Human Lung Cryo-sections of human lung tissue were incubated with 20 μg/ml of purified DENV-2 NS1 or BSA for 1 h at room temperature. Bound NS1 was detected by staining with a mixture of DENV NS1 mAbs (1A4, 1F11, 2G6, 1B2) followed by Alexa Fluor 488 conjugated with goat anti-mouse IgG. Subsequently, the sections were co-stained with rabbit anti-human CD31 (PECAM-1) followed by Cy3-conjugated donkey anti-rabbit IgG. Nuclei were stained with a DNA-specific dye (Hoechst) and the sections were analyzed by confocal microscopy. Sections incubated with purified DENV NS1 and stained with isotype mAbs served as a negative control.

    Journal: PLoS Pathogens

    Article Title: Secreted NS1 of Dengue Virus Attaches to the Surface of Cells via Interactions with Heparan Sulfate and Chondroitin Sulfate E

    doi: 10.1371/journal.ppat.0030183

    Figure Lengend Snippet: Binding of DENV NS1 to Human Lung Cryo-sections of human lung tissue were incubated with 20 μg/ml of purified DENV-2 NS1 or BSA for 1 h at room temperature. Bound NS1 was detected by staining with a mixture of DENV NS1 mAbs (1A4, 1F11, 2G6, 1B2) followed by Alexa Fluor 488 conjugated with goat anti-mouse IgG. Subsequently, the sections were co-stained with rabbit anti-human CD31 (PECAM-1) followed by Cy3-conjugated donkey anti-rabbit IgG. Nuclei were stained with a DNA-specific dye (Hoechst) and the sections were analyzed by confocal microscopy. Sections incubated with purified DENV NS1 and stained with isotype mAbs served as a negative control.

    Article Snippet: After three washes, sections were incubated with Alexa Fluor 488 conjugated goat anti-mouse IgG secondary antibody (Invitrogen) and followed by 1-h incubation at room temperature with rabbit anti-human CD31 (PECAM) (Santa Cruz Biotechnology) at the dilution of 1:10.

    Techniques: Binding Assay, Incubation, Purification, Staining, Confocal Microscopy, Negative Control

    Direct injection results in elevated S1P levels which correlate with the activation of receptor 1 in muscle fibers. ( A ) To quantify the elevation of S1P following direct administration, we injected a single dose (same dose as Figure 5 ) of S1P in left TAs and vehicle in right TAs of uninjured mdx 4cv (n = 3, 11-MO) mice. TA muscles were harvested 15 minutes post injection for analysis by LC-MS/MS. Results indicate a significant elevation of S1P following direct injection. ( B ) To visualize the location of S1P following injection, biotinylated-S1P was injected in left TAs versus vehicle in right TAs of uninjured mdx 4cv mice (n = 2, 11-MO). Once more, TAs were harvested 15 minutes following injection. Staining with streptavidin conjugated to Alexa Fluor 594 reveals the presence of S1P-biotin around the perimeter of muscle fibers. ( C ) Staining of mdx 4cv TAs for S1PR1 and S1PR3 reveals S1PR1 is localized to the perimeter and perinuclear area (arrow) of muscle fibers (left photo). In contrast, staining for S1PR3 was mainly localized to the muscle vasculature (middle photo). Staining in parallel with an IgG isotype control for both antibodies shows the absence of non-specific staining (right graph). ( D ) Staining for S1PR1 in CTX-injured TAs (same tissue from Figure 5 ) reveals S1PR1 is present at the perimeter and perinuclear area of regenerating eMyHC+ fibers. ( E ) Staining for phosphorylated S1PR1 in the same mdx 4cv TAs was more prominent in the perinuclear area of eMyHC+ fibers, indicating the presence of active S1PR1 signaling in regenerating fibers. Scale bars = 50 μm. ** P

    Journal: Skeletal Muscle

    Article Title: Increased sphingosine-1-phosphate improves muscle regeneration in acutely injured mdx mice

    doi: 10.1186/2044-5040-3-20

    Figure Lengend Snippet: Direct injection results in elevated S1P levels which correlate with the activation of receptor 1 in muscle fibers. ( A ) To quantify the elevation of S1P following direct administration, we injected a single dose (same dose as Figure 5 ) of S1P in left TAs and vehicle in right TAs of uninjured mdx 4cv (n = 3, 11-MO) mice. TA muscles were harvested 15 minutes post injection for analysis by LC-MS/MS. Results indicate a significant elevation of S1P following direct injection. ( B ) To visualize the location of S1P following injection, biotinylated-S1P was injected in left TAs versus vehicle in right TAs of uninjured mdx 4cv mice (n = 2, 11-MO). Once more, TAs were harvested 15 minutes following injection. Staining with streptavidin conjugated to Alexa Fluor 594 reveals the presence of S1P-biotin around the perimeter of muscle fibers. ( C ) Staining of mdx 4cv TAs for S1PR1 and S1PR3 reveals S1PR1 is localized to the perimeter and perinuclear area (arrow) of muscle fibers (left photo). In contrast, staining for S1PR3 was mainly localized to the muscle vasculature (middle photo). Staining in parallel with an IgG isotype control for both antibodies shows the absence of non-specific staining (right graph). ( D ) Staining for S1PR1 in CTX-injured TAs (same tissue from Figure 5 ) reveals S1PR1 is present at the perimeter and perinuclear area of regenerating eMyHC+ fibers. ( E ) Staining for phosphorylated S1PR1 in the same mdx 4cv TAs was more prominent in the perinuclear area of eMyHC+ fibers, indicating the presence of active S1PR1 signaling in regenerating fibers. Scale bars = 50 μm. ** P

    Article Snippet: Following washes, Alexa Fluor 488 conjugated goat anti-rabbit IgG (Life Technologies) was administered at 1:800 dilution for 1 hour.

    Techniques: Injection, Activation Assay, Mouse Assay, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Staining