alexa 594 goat anti rabbit igg  (Thermo Fisher)


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    Structured Review

    Thermo Fisher alexa 594 goat anti rabbit igg
    Immunophenotype and functional analysis of cultured FACS sorted cardiac endothelial cells. The upper panel were representative photograph of immunofluorescent staining of cultured FACS sorted cardiac endothelial cells for antiCaveolin-1, anti-eNOS, and anti-vWF followed by anti rabbit <t>IgG</t> Alexa 594. Staining with only secondary anti rabbit IgG <t>Alexa</t> 594 antibody shows minimal background red fluorescence. Left panel shows the monochromic photograph for each antibody. Right panel shows each antibody in red and DAPI in blue. Scale bar = 100 μ m. The lower panel were cultured FACS sorted cardiac endothelial cells up took Ac-LDL-rhodamine (red), also stained with DAPI (blue) and cultured FACS sorted cardiac endothelial cells were plated in Matrigel for the formation of capillary-like structures. Photographs were taken at 3 hours (scale bar = 500 μ m) and 5.5 hours (scale bar = 100 μ m) after incubation.
    Alexa 594 Goat Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 408 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexa 594 goat anti rabbit igg/product/Thermo Fisher
    Average 89 stars, based on 408 article reviews
    Price from $9.99 to $1999.99
    alexa 594 goat anti rabbit igg - by Bioz Stars, 2020-07
    89/100 stars

    Images

    1) Product Images from "Isolation, Characterization, and Transplantation of Cardiac Endothelial Cells"

    Article Title: Isolation, Characterization, and Transplantation of Cardiac Endothelial Cells

    Journal: BioMed Research International

    doi: 10.1155/2013/359412

    Immunophenotype and functional analysis of cultured FACS sorted cardiac endothelial cells. The upper panel were representative photograph of immunofluorescent staining of cultured FACS sorted cardiac endothelial cells for antiCaveolin-1, anti-eNOS, and anti-vWF followed by anti rabbit IgG Alexa 594. Staining with only secondary anti rabbit IgG Alexa 594 antibody shows minimal background red fluorescence. Left panel shows the monochromic photograph for each antibody. Right panel shows each antibody in red and DAPI in blue. Scale bar = 100 μ m. The lower panel were cultured FACS sorted cardiac endothelial cells up took Ac-LDL-rhodamine (red), also stained with DAPI (blue) and cultured FACS sorted cardiac endothelial cells were plated in Matrigel for the formation of capillary-like structures. Photographs were taken at 3 hours (scale bar = 500 μ m) and 5.5 hours (scale bar = 100 μ m) after incubation.
    Figure Legend Snippet: Immunophenotype and functional analysis of cultured FACS sorted cardiac endothelial cells. The upper panel were representative photograph of immunofluorescent staining of cultured FACS sorted cardiac endothelial cells for antiCaveolin-1, anti-eNOS, and anti-vWF followed by anti rabbit IgG Alexa 594. Staining with only secondary anti rabbit IgG Alexa 594 antibody shows minimal background red fluorescence. Left panel shows the monochromic photograph for each antibody. Right panel shows each antibody in red and DAPI in blue. Scale bar = 100 μ m. The lower panel were cultured FACS sorted cardiac endothelial cells up took Ac-LDL-rhodamine (red), also stained with DAPI (blue) and cultured FACS sorted cardiac endothelial cells were plated in Matrigel for the formation of capillary-like structures. Photographs were taken at 3 hours (scale bar = 500 μ m) and 5.5 hours (scale bar = 100 μ m) after incubation.

    Techniques Used: Functional Assay, Cell Culture, FACS, Staining, Fluorescence, Incubation

    2) Product Images from "Complement-Related Proteins Control the Flavivirus Infection of Aedes aegypti by Inducing Antimicrobial Peptides"

    Article Title: Complement-Related Proteins Control the Flavivirus Infection of Aedes aegypti by Inducing Antimicrobial Peptides

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004027

    A Scavenger receptor-C with CCP domains recognizes DENV-2 in vitro and in vivo . (A) Percentage of amino acid identity of insect SR-Cs. (B) Schematic representation of SR-Cs in A. aegypti and D. melanogaster . The functional modules were predicted in SMART ( http://smart.embl-heidelberg.de/smart/set_mode.cgi?GENOMIC=1 ) and Pfam websites ( http://pfam.sanger.ac.uk/ ). (C) Expression and purification of AaSR-C from Drosophila S2 cells. The extracellular region (AaSR-C-Ex) or full length (AaSR-C-Full) AaSR-C was cloned into the pMT/BiP/V5-His-A expression vector. The recombinant plasmids were transfected into Drosophila S2 cells, and their expression was probed using an anti-V5 mAb. The supernatant or lysates from mock-transfected S2 cells was used as the mock control (Left panel). Recombinant AaSR-C-Ex, produced in Drosophila cells, was purified using an Ni-His column (Right panel). (D) AaSR-C-Ex interacted with DENV-2 E proteins in co-immunoprecipitation assay. Purified AaSR-C-Ex (V5) and DENV-2 E (FLAG) were used to investigate the interaction of the proteins. Control rabbit IgG was used as a mock control to exclude non-specific interactions. The protein complex was pulled down with a rabbit anti-FLAG antibody and detected using a mouse anti-V5 antibody. We reproduced the experiment 3 times. (E) AaSR-C-Ex captured DENV-2 virions in an ELISA. Binding was probed using the flavivirus E mAb 4G2. The data are presented as the mean ± standard error. The experiment was reproduced 3 times. (F) AaSR-C bound DENV-2 virions on the cell surface. A Cu 2+ -inducible stable S2 cell line was generated to express the full-length AaSR-C. DENV-2 virions were incubated with AaSR-C expressing cells at 4°C for 1 hr. Non-induced stable cells and empty vector-transfected S2 cells containing virions served as the mock control groups. The interaction between AaSR-C and DENV was measured through flow cytometry. The DENV virions were stained using the flaviviral E mAb 4G2 and anti-mouse IgG Alexa-488; AaSR-C was probed using a Myc mAb and anti-rabbit IgG Phycoerythrin (PE). The data was analyzed using FlowJo software. The presented data was representative of 3 independent experiments with similar results. (G) The in vivo association between AaSR-C and DENV-2 in A.aegypti hemocytes. Hemolymph was collected from uninfected mosquitoes, AaSR-C silenced infected mosquitoes and GFP dsRNA treated infected mosquitoes to undergo immunofluorescence staining. AaSR-C was stained with anti-rabbit IgG Alexa-488 (Green), and the DENV-2 E protein was identified using anti-mouse IgG Alexa-546 (Red). Nuclei were stained blue with To-Pro-3 iodide (Blue). Images were examined using a Zeiss LSM 780 meta confocal 63×objective lens.
    Figure Legend Snippet: A Scavenger receptor-C with CCP domains recognizes DENV-2 in vitro and in vivo . (A) Percentage of amino acid identity of insect SR-Cs. (B) Schematic representation of SR-Cs in A. aegypti and D. melanogaster . The functional modules were predicted in SMART ( http://smart.embl-heidelberg.de/smart/set_mode.cgi?GENOMIC=1 ) and Pfam websites ( http://pfam.sanger.ac.uk/ ). (C) Expression and purification of AaSR-C from Drosophila S2 cells. The extracellular region (AaSR-C-Ex) or full length (AaSR-C-Full) AaSR-C was cloned into the pMT/BiP/V5-His-A expression vector. The recombinant plasmids were transfected into Drosophila S2 cells, and their expression was probed using an anti-V5 mAb. The supernatant or lysates from mock-transfected S2 cells was used as the mock control (Left panel). Recombinant AaSR-C-Ex, produced in Drosophila cells, was purified using an Ni-His column (Right panel). (D) AaSR-C-Ex interacted with DENV-2 E proteins in co-immunoprecipitation assay. Purified AaSR-C-Ex (V5) and DENV-2 E (FLAG) were used to investigate the interaction of the proteins. Control rabbit IgG was used as a mock control to exclude non-specific interactions. The protein complex was pulled down with a rabbit anti-FLAG antibody and detected using a mouse anti-V5 antibody. We reproduced the experiment 3 times. (E) AaSR-C-Ex captured DENV-2 virions in an ELISA. Binding was probed using the flavivirus E mAb 4G2. The data are presented as the mean ± standard error. The experiment was reproduced 3 times. (F) AaSR-C bound DENV-2 virions on the cell surface. A Cu 2+ -inducible stable S2 cell line was generated to express the full-length AaSR-C. DENV-2 virions were incubated with AaSR-C expressing cells at 4°C for 1 hr. Non-induced stable cells and empty vector-transfected S2 cells containing virions served as the mock control groups. The interaction between AaSR-C and DENV was measured through flow cytometry. The DENV virions were stained using the flaviviral E mAb 4G2 and anti-mouse IgG Alexa-488; AaSR-C was probed using a Myc mAb and anti-rabbit IgG Phycoerythrin (PE). The data was analyzed using FlowJo software. The presented data was representative of 3 independent experiments with similar results. (G) The in vivo association between AaSR-C and DENV-2 in A.aegypti hemocytes. Hemolymph was collected from uninfected mosquitoes, AaSR-C silenced infected mosquitoes and GFP dsRNA treated infected mosquitoes to undergo immunofluorescence staining. AaSR-C was stained with anti-rabbit IgG Alexa-488 (Green), and the DENV-2 E protein was identified using anti-mouse IgG Alexa-546 (Red). Nuclei were stained blue with To-Pro-3 iodide (Blue). Images were examined using a Zeiss LSM 780 meta confocal 63×objective lens.

    Techniques Used: In Vitro, In Vivo, Functional Assay, Expressing, Purification, Clone Assay, Plasmid Preparation, Recombinant, Transfection, Produced, Co-Immunoprecipitation Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Generated, Incubation, Flow Cytometry, Cytometry, Staining, Software, Infection, Immunofluorescence

    AaMCR and AaSR-C function in a pathway that opposes DENV-2 infection. (A) The interaction between 3 AaMCR fragments and AaSR-C in co-IP assays. Three AaMCR gene fragments were cloned into the pMT/BiP/V5-His A vector. The recombinant plasmids were transiently transfected into S2 cells. The cell supernatant was used for investigation of the AaMCR/AaSR-C interaction. The protein complex was pulled down with a rabbit anti-V5 antibody and probed using a mouse anti-HA antibody. The experiment was reproduced 3 times. (B) Expression and purification of AaMCR-a in Drosophila S2 cells. The purified AaMCR-a was separated through SDS-PAGE (Left Panel) and detected with an anti-HA antibody via western blotting (Right Panel). The supernatant from empty vector-transfected S2 cells was used as the mock control. (C) AaSR-C-Ex acted as an adaptor in the interaction between the AaMCR-a and DENV-2 E proteins. The purified AaSR-C-Ex, AaMCR-a and DENV-2 E proteins were mixed and pulled down with a mouse anti-HA antibody (AaMCR-a) and detected using a rabbit anti-V5 antibody (AaSR-C) and anti-FLAG-HRP antibody (DENV-2 E). The experiment was repeated 3 times with similar results. (D) AaSR-C-Ex connected AaMCR-a to DENV-2 virions. Purified AaMCR-a or BSA was pre-coated into the ELISA plate wells. DENV-2 virions either mixed with AaSR-C-Ex or without AaSR-C-Ex were added to the protein-coated wells. The signal was detected using the flavivirus E mAb 4G2. The data are expressed as the mean ± standard error. The experiment was reproduced by 3 times with similar results. (E) Double knockdown of AaMCR and AaSR-C showed similar effects on DENV-2 infection to individual knockdown. Both AaSR-C (i) and AaMCR (ii) were knocked down using a dsRNA mixture in the AaSR-C / AaMCR co-silenced group. DENV-2 replication and the numbers of infectious DENV-2 virions in the mosquitoes were measured via qPCR (iii) and plaque assays (iv). Statistical analysis was performed using the non-parametric Mann-Whitney test. The data on gene silencing (i, ii) and from plaque assays (iv) are expressed as the mean ± standard error. The horizontal line depicts the median (iii). Each dot represents an individual mosquito. The result was representative of 3 independent experiments. (F) Immunostaining of AaMCR, AaSR-C and DENV-2 in A. aegypti hemocytes. The hemocytes were dissected from uninfected mosquitoes, AaMCR and/or AaSR-C silenced infected mosquitoes and GFP dsRNA treated infected mosquitoes at 6 days post-infection. AaSR-C was stained with anti-rabbit IgG Alexa-488 (Green); AaMCR was probed using anti-mouse IgG Alexa-546 (Red); the DENV-2 E protein was probed with DENV-2 human antiserum (purified IgG) and anti-human IgG Alexa-633 (Blue). Images were examined using a Zeiss LSM 780 meta confocal 63×objective lens.
    Figure Legend Snippet: AaMCR and AaSR-C function in a pathway that opposes DENV-2 infection. (A) The interaction between 3 AaMCR fragments and AaSR-C in co-IP assays. Three AaMCR gene fragments were cloned into the pMT/BiP/V5-His A vector. The recombinant plasmids were transiently transfected into S2 cells. The cell supernatant was used for investigation of the AaMCR/AaSR-C interaction. The protein complex was pulled down with a rabbit anti-V5 antibody and probed using a mouse anti-HA antibody. The experiment was reproduced 3 times. (B) Expression and purification of AaMCR-a in Drosophila S2 cells. The purified AaMCR-a was separated through SDS-PAGE (Left Panel) and detected with an anti-HA antibody via western blotting (Right Panel). The supernatant from empty vector-transfected S2 cells was used as the mock control. (C) AaSR-C-Ex acted as an adaptor in the interaction between the AaMCR-a and DENV-2 E proteins. The purified AaSR-C-Ex, AaMCR-a and DENV-2 E proteins were mixed and pulled down with a mouse anti-HA antibody (AaMCR-a) and detected using a rabbit anti-V5 antibody (AaSR-C) and anti-FLAG-HRP antibody (DENV-2 E). The experiment was repeated 3 times with similar results. (D) AaSR-C-Ex connected AaMCR-a to DENV-2 virions. Purified AaMCR-a or BSA was pre-coated into the ELISA plate wells. DENV-2 virions either mixed with AaSR-C-Ex or without AaSR-C-Ex were added to the protein-coated wells. The signal was detected using the flavivirus E mAb 4G2. The data are expressed as the mean ± standard error. The experiment was reproduced by 3 times with similar results. (E) Double knockdown of AaMCR and AaSR-C showed similar effects on DENV-2 infection to individual knockdown. Both AaSR-C (i) and AaMCR (ii) were knocked down using a dsRNA mixture in the AaSR-C / AaMCR co-silenced group. DENV-2 replication and the numbers of infectious DENV-2 virions in the mosquitoes were measured via qPCR (iii) and plaque assays (iv). Statistical analysis was performed using the non-parametric Mann-Whitney test. The data on gene silencing (i, ii) and from plaque assays (iv) are expressed as the mean ± standard error. The horizontal line depicts the median (iii). Each dot represents an individual mosquito. The result was representative of 3 independent experiments. (F) Immunostaining of AaMCR, AaSR-C and DENV-2 in A. aegypti hemocytes. The hemocytes were dissected from uninfected mosquitoes, AaMCR and/or AaSR-C silenced infected mosquitoes and GFP dsRNA treated infected mosquitoes at 6 days post-infection. AaSR-C was stained with anti-rabbit IgG Alexa-488 (Green); AaMCR was probed using anti-mouse IgG Alexa-546 (Red); the DENV-2 E protein was probed with DENV-2 human antiserum (purified IgG) and anti-human IgG Alexa-633 (Blue). Images were examined using a Zeiss LSM 780 meta confocal 63×objective lens.

    Techniques Used: Infection, Co-Immunoprecipitation Assay, Clone Assay, Plasmid Preparation, Recombinant, Transfection, Expressing, Purification, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, MANN-WHITNEY, Immunostaining, Staining

    3) Product Images from "Intranodal vaccination with mRNA-optimized dendritic cells in metastatic melanoma patients"

    Article Title: Intranodal vaccination with mRNA-optimized dendritic cells in metastatic melanoma patients

    Journal: Oncoimmunology

    doi: 10.1080/2162402X.2015.1019197

    KLH-specific immune responses before and after dendritic cell (DC) vaccination. KLH-specific T cell proliferation was analyzed after each DC vaccination during the first vaccination cycle in peripheral blood mononuclear cells of melanoma patients. The proliferative response to KLH is given as a proliferation index (proliferation with KLH/proliferation without KLH) ( A ). KLH-specific IgG ( B ), IgA ( C ) and IgM antibodies ( D ) were quantitatively measured before and after each vaccination cycle in sera of vaccinated patients. The best Ig response was shown for each patient. Each dot represents 1 patient or the number of patients as indicated.
    Figure Legend Snippet: KLH-specific immune responses before and after dendritic cell (DC) vaccination. KLH-specific T cell proliferation was analyzed after each DC vaccination during the first vaccination cycle in peripheral blood mononuclear cells of melanoma patients. The proliferative response to KLH is given as a proliferation index (proliferation with KLH/proliferation without KLH) ( A ). KLH-specific IgG ( B ), IgA ( C ) and IgM antibodies ( D ) were quantitatively measured before and after each vaccination cycle in sera of vaccinated patients. The best Ig response was shown for each patient. Each dot represents 1 patient or the number of patients as indicated.

    Techniques Used:

    4) Product Images from "Use of short interfering RNA delivered by cationic liposomes to enable efficient down-regulation of PTPN22 gene in human T lymphocytes"

    Article Title: Use of short interfering RNA delivered by cationic liposomes to enable efficient down-regulation of PTPN22 gene in human T lymphocytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0175784

    Confocal microscopy analysis of Lyp protein expression in Jurkat T cells after transfection with lipoplexes. The comparison shown is between samples transfected with 60 and 100 pmols of Lipo/siRNA lipoplexes, and analyzed after 72 hours from the beginning of the O/N transfection. Control cells are untreated and cultured in RPMI (upper panels). Lyp protein expression is revealed by anti-mouse IgG conjugated to Alexa Fluor 555 (red signal). Cell morphology and DNA are detected by WGA (green) and Hoechst (blue) staining, respectively. Bar: 20 μm.
    Figure Legend Snippet: Confocal microscopy analysis of Lyp protein expression in Jurkat T cells after transfection with lipoplexes. The comparison shown is between samples transfected with 60 and 100 pmols of Lipo/siRNA lipoplexes, and analyzed after 72 hours from the beginning of the O/N transfection. Control cells are untreated and cultured in RPMI (upper panels). Lyp protein expression is revealed by anti-mouse IgG conjugated to Alexa Fluor 555 (red signal). Cell morphology and DNA are detected by WGA (green) and Hoechst (blue) staining, respectively. Bar: 20 μm.

    Techniques Used: Confocal Microscopy, Expressing, Transfection, Cell Culture, Whole Genome Amplification, Staining

    5) Product Images from "Novel Therapy for Atherosclerosis Using Recombinant Immunotoxin Against Folate Receptor ?-Expressing Macrophages"

    Article Title: Novel Therapy for Atherosclerosis Using Recombinant Immunotoxin Against Folate Receptor ?-Expressing Macrophages

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

    doi: 10.1161/JAHA.112.003079

    Representative double fluorescence immunohistochemistry of aortic cross sections at 21 wk of age. Representative photographs of FRβ-expressing cells in atherosclerotic lesions. Cryosectioned aortic sinus lesions were stained with (A) anti-CD68 (green) and anti-FRβ followed by Alexa Fluor 594 (red), (B) anti-αSMA (green) and FRβ (red), (C) anti-CD31 (green) and FRβ (red), or (D) anti-CD3 (green) and FRβ (red). FRβ was colocalized with CD68 but not with CD31, α-SMA, or CD3 (scale bar=50 μm). FRβ, folate receptor-β; α-SMA, α-smooth muscle actin.
    Figure Legend Snippet: Representative double fluorescence immunohistochemistry of aortic cross sections at 21 wk of age. Representative photographs of FRβ-expressing cells in atherosclerotic lesions. Cryosectioned aortic sinus lesions were stained with (A) anti-CD68 (green) and anti-FRβ followed by Alexa Fluor 594 (red), (B) anti-αSMA (green) and FRβ (red), (C) anti-CD31 (green) and FRβ (red), or (D) anti-CD3 (green) and FRβ (red). FRβ was colocalized with CD68 but not with CD31, α-SMA, or CD3 (scale bar=50 μm). FRβ, folate receptor-β; α-SMA, α-smooth muscle actin.

    Techniques Used: Fluorescence, Immunohistochemistry, Expressing, Staining

    6) Product Images from "Galactose 6-O-Sulfotransferases Are Not Required for the Generation of Siglec-F Ligands in Leukocytes or Lung Tissue *"

    Article Title: Galactose 6-O-Sulfotransferases Are Not Required for the Generation of Siglec-F Ligands in Leukocytes or Lung Tissue *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.485409

    Siglec-F ligand expression in peripheral blood leukocytes. A, flow cytometry analysis of leukocyte subsets stained with Siglec-F-Fc, Siglec-E-Fc, and CD22-Fc ( red histograms ). Staining after sialidase treatment ( black histograms ) and staining with human IgG ( gray histograms ) are shown. Results are representative of four independent experiments. B, flow cytometry analysis of Siglec-F expression on leukocytes from wild type ( black histograms ) or Siglec-F KO mice ( gray histograms ). Results are representative of two independent experiments. Eos , eosinophils; Neut , neutrophils; Baso , basophils; Mono , classical monocytes; NK , natural killer cells; T , T cells; B , B cells; Alv Mac , alveolar macrophages.
    Figure Legend Snippet: Siglec-F ligand expression in peripheral blood leukocytes. A, flow cytometry analysis of leukocyte subsets stained with Siglec-F-Fc, Siglec-E-Fc, and CD22-Fc ( red histograms ). Staining after sialidase treatment ( black histograms ) and staining with human IgG ( gray histograms ) are shown. Results are representative of four independent experiments. B, flow cytometry analysis of Siglec-F expression on leukocytes from wild type ( black histograms ) or Siglec-F KO mice ( gray histograms ). Results are representative of two independent experiments. Eos , eosinophils; Neut , neutrophils; Baso , basophils; Mono , classical monocytes; NK , natural killer cells; T , T cells; B , B cells; Alv Mac , alveolar macrophages.

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Staining, Mouse Assay

    MS analysis of sulfated glycans in eosinophils. A , flow cytometry analysis of IL-5 transgenic eosinophils stained with Siglec-F-Fc ( red histogram ). Staining after sialidase treatment ( black histogram ) and staining with human IgG ( gray histogram ) is shown. Results are representative of three independent experiments. B, extracted ion chromatograms of the major sulfated O- glycans from IL-5 transgenic eosinophils as detected by nanoLC-MS/MS analysis. The m / z values for the [M − H] − molecular ions afforded by the mono-sulfated permethylated O- glycans were annotated along with the assigned structures based on interpretation of the HCD and CID MS/MS data. The relative peak heights are indicative of the relative abundance of each of the sulfated, sialylated core 1 and core 2 O- glycans. C, low mass regions of the negative ion mode nanoESI HCD and CID MS/MS spectra of mono-sulfated di-sialylated ( left ) and mono-sulfated mono-sialylated ( right ) structures. Assignment of the major peaks for all spectra is annotated using the standard schematic symbols. Eos , eosinophils.
    Figure Legend Snippet: MS analysis of sulfated glycans in eosinophils. A , flow cytometry analysis of IL-5 transgenic eosinophils stained with Siglec-F-Fc ( red histogram ). Staining after sialidase treatment ( black histogram ) and staining with human IgG ( gray histogram ) is shown. Results are representative of three independent experiments. B, extracted ion chromatograms of the major sulfated O- glycans from IL-5 transgenic eosinophils as detected by nanoLC-MS/MS analysis. The m / z values for the [M − H] − molecular ions afforded by the mono-sulfated permethylated O- glycans were annotated along with the assigned structures based on interpretation of the HCD and CID MS/MS data. The relative peak heights are indicative of the relative abundance of each of the sulfated, sialylated core 1 and core 2 O- glycans. C, low mass regions of the negative ion mode nanoESI HCD and CID MS/MS spectra of mono-sulfated di-sialylated ( left ) and mono-sulfated mono-sialylated ( right ) structures. Assignment of the major peaks for all spectra is annotated using the standard schematic symbols. Eos , eosinophils.

    Techniques Used: Mass Spectrometry, Flow Cytometry, Cytometry, Transgenic Assay, Staining

    Siglec-F ligand expression in leukocytes from KSGal6ST/C6ST-1 DKO mice. A, flow cytometry analysis of leukocyte subsets stained with Siglec-F-Fc ( red histograms ). Staining after sialidase treatment ( black histograms ) and staining with human IgG ( gray histograms ) are shown. Scale bars represent 50 μm. Results are representative of two independent experiments. B, cryostat-cut sections of lungs from N. brasiliensis -infected mice. Sections were stained with Siglec-F-Fc ( green ), anti-eMBP ( red ), anti-proSP-C ( blue ), and DAPI ( white ). Low power ( top ) and high power ( bottom ) fields are shown. Eos , eosinophils; Neut , neutrophils; Mono , classical monocytes; NK , natural killer cells.
    Figure Legend Snippet: Siglec-F ligand expression in leukocytes from KSGal6ST/C6ST-1 DKO mice. A, flow cytometry analysis of leukocyte subsets stained with Siglec-F-Fc ( red histograms ). Staining after sialidase treatment ( black histograms ) and staining with human IgG ( gray histograms ) are shown. Scale bars represent 50 μm. Results are representative of two independent experiments. B, cryostat-cut sections of lungs from N. brasiliensis -infected mice. Sections were stained with Siglec-F-Fc ( green ), anti-eMBP ( red ), anti-proSP-C ( blue ), and DAPI ( white ). Low power ( top ) and high power ( bottom ) fields are shown. Eos , eosinophils; Neut , neutrophils; Mono , classical monocytes; NK , natural killer cells.

    Techniques Used: Expressing, Mouse Assay, Flow Cytometry, Cytometry, Staining, Infection

    Siglec-F ligands in BAL fluid. A, fractions of BAL fluid assayed by ELISA using Siglec-F-Fc ( filled circles ), or anti-MUC5B ( filled squares ). Total protein was determined by measuring absorbance at 280 nm ( dotted line ). No signal was observed when wells were reacted with CD22-Fc ( open squares ), human IgG ( open triangles ), or treated with sialidase before incubation with Siglec-F-Fc ( open circles ). Results are representative of two independent experiments.
    Figure Legend Snippet: Siglec-F ligands in BAL fluid. A, fractions of BAL fluid assayed by ELISA using Siglec-F-Fc ( filled circles ), or anti-MUC5B ( filled squares ). Total protein was determined by measuring absorbance at 280 nm ( dotted line ). No signal was observed when wells were reacted with CD22-Fc ( open squares ), human IgG ( open triangles ), or treated with sialidase before incubation with Siglec-F-Fc ( open circles ). Results are representative of two independent experiments.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Incubation

    7) Product Images from "Tetherin Antagonism by HIV-1 Group M Nef Proteins"

    Article Title: Tetherin Antagonism by HIV-1 Group M Nef Proteins

    Journal: Journal of Virology

    doi: 10.1128/JVI.01465-16

    Tetherin antagonism by Nef protects HIV-1-infected cells from ADCC. (A) CEM.NKR- CCR5 -sLTR-Luc cells were infected with wild-type HIV-1 NL4-3 (Vpu + ), HIV-1 NL4-3 Δ vpu , or HIV-1.Δ vpu .IeG-Nef recombinants expressing the indicated Nef alleles and incubated with a CD16 + NK cell line at an effector-to-target cell ratio of 10:1 in the presence of serial dilutions of purified IgG from HIV-1-positive donors (HIVIG). ADCC was calculated from the luciferase activity (RLU) after an 8-h incubation. Error bars indicate the standard deviations of the means for triplicate wells at each antibody concentration, and the dotted line indicates 50% killing of HIV-1-infected cells. (B) Nef and p55 Gag expression in virus-infected cells was confirmed by Western blot analysis of cell lysates with staining for β-actin as a control for sample loading. (C and D) Histograms showing the fluorescence intensity of tetherin (BST-2) (C) and Env (D) staining on the surface of viable, HIV-1-infected (p55 + CD4 low ) cells as described above for panel A. Tetherin and Env staining on cells infected with virus expressing NL4-3 Nef is shown as a reference (red histograms). The shaded histograms indicate nonspecific staining with either an isotype control for the BST-2-specific antibody (C) or IgG from HIV-negative donors (D). (E and F) Susceptibility to ADCC correlates with the fluorescence intensity of tetherin (E) and Env (F) staining on the surface of virus-infected cells (Pearson correlation test). The data are representative of results from three independent experiments.
    Figure Legend Snippet: Tetherin antagonism by Nef protects HIV-1-infected cells from ADCC. (A) CEM.NKR- CCR5 -sLTR-Luc cells were infected with wild-type HIV-1 NL4-3 (Vpu + ), HIV-1 NL4-3 Δ vpu , or HIV-1.Δ vpu .IeG-Nef recombinants expressing the indicated Nef alleles and incubated with a CD16 + NK cell line at an effector-to-target cell ratio of 10:1 in the presence of serial dilutions of purified IgG from HIV-1-positive donors (HIVIG). ADCC was calculated from the luciferase activity (RLU) after an 8-h incubation. Error bars indicate the standard deviations of the means for triplicate wells at each antibody concentration, and the dotted line indicates 50% killing of HIV-1-infected cells. (B) Nef and p55 Gag expression in virus-infected cells was confirmed by Western blot analysis of cell lysates with staining for β-actin as a control for sample loading. (C and D) Histograms showing the fluorescence intensity of tetherin (BST-2) (C) and Env (D) staining on the surface of viable, HIV-1-infected (p55 + CD4 low ) cells as described above for panel A. Tetherin and Env staining on cells infected with virus expressing NL4-3 Nef is shown as a reference (red histograms). The shaded histograms indicate nonspecific staining with either an isotype control for the BST-2-specific antibody (C) or IgG from HIV-negative donors (D). (E and F) Susceptibility to ADCC correlates with the fluorescence intensity of tetherin (E) and Env (F) staining on the surface of virus-infected cells (Pearson correlation test). The data are representative of results from three independent experiments.

    Techniques Used: Infection, Expressing, Incubation, Purification, Luciferase, Activity Assay, Concentration Assay, Western Blot, Staining, Fluorescence

    8) Product Images from "Axin expression in thymic stromal cells contributes to an age-related increase in thymic adiposity and is associated with reduced thymopoiesis independently of ghrelin signaling"

    Article Title: Axin expression in thymic stromal cells contributes to an age-related increase in thymic adiposity and is associated with reduced thymopoiesis independently of ghrelin signaling

    Journal: Journal of Leukocyte Biology

    doi: 10.1189/jlb.1008621

    Age-related increase in axin-expressing TF is blocked by CR. (A) TF were stained with ERTR7, followed by specific secondary antibody labeled with Alexa Fluor 488 (green), and axin was visualized by Alexa Fluor 594 (red)-conjugated antibody. The elevated axin expression in 12-month-old thymus colocalized with ERTR7. Representative images (original, 200×) of thymic sections of four mice in each group are shown. (B) Axin also partially colocalizes with FSP-1-expressing TF. The negative isotype IgG control antibody displayed no specific staining in the negative control cryosections of young and old mice.
    Figure Legend Snippet: Age-related increase in axin-expressing TF is blocked by CR. (A) TF were stained with ERTR7, followed by specific secondary antibody labeled with Alexa Fluor 488 (green), and axin was visualized by Alexa Fluor 594 (red)-conjugated antibody. The elevated axin expression in 12-month-old thymus colocalized with ERTR7. Representative images (original, 200×) of thymic sections of four mice in each group are shown. (B) Axin also partially colocalizes with FSP-1-expressing TF. The negative isotype IgG control antibody displayed no specific staining in the negative control cryosections of young and old mice.

    Techniques Used: Expressing, Staining, Labeling, Mouse Assay, Negative Control

    9) Product Images from "The Membrane-Proximal Region of C–C Chemokine Receptor Type 5 Participates in the Infection of HIV-1"

    Article Title: The Membrane-Proximal Region of C–C Chemokine Receptor Type 5 Participates in the Infection of HIV-1

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.00478

    Inducing membrane-proximal region (MPR)-specific antibodies in mice . (A) C–C chemokine receptor type 5 (CCR5) consists of an N-terminal, three extracellular loops (ECLs), three intracellular loops, and a C-terminal tail. The structure of CCR5 was rendered by Pymol software based on a previously reported structure 4MBS ( 7 ). MPR and ECL2 are highlighted in blue and green, respectively. ECL2 contains a binding site for 2D7. (B) The amino acid sequence of N-terminal domain and first transmembrane helix (TM1) of CCR5. The antigenic regions were predicted by SVMTrip, PAP, and FBCPred methods and shown under the sequence of CCR5. (C) Balb/c mice were immunized with one dose of C17-keyhole limpet hemocyanin (KLH) plus complete Freund’s adjuvant, one dose of C17-KLH plus incomplete Freund’s adjuvant (IFA), and two doses of a mixture of C17-KLH, C17 peptide, and IFA. Sera were collected 1 week after the last immunization and antibody titers against C17-KLH, C17, or an irrelevant C34 peptide were measured by enzyme-linked immunosorbent assay. n = 3. (D) Total IgGs were purified from antiserum, designated as anti-C17. The binding of anti-C17 to CCR5 expressed on the cell surface of Ghost-R5 cells was determined by flow cytometry. Normal IgGs purified from the sera of un-immunized mice served as a negative control, while 2D7 served as a positive control. Alexa Fluor 647-conjugated anti-mouse IgG was used as the secondary antibody. C17 peptide and an irrelevant C34 peptide were used to confirm the binding specificity. Data are presented as mean ± SEM. All experiments were repeated twice with similar results.
    Figure Legend Snippet: Inducing membrane-proximal region (MPR)-specific antibodies in mice . (A) C–C chemokine receptor type 5 (CCR5) consists of an N-terminal, three extracellular loops (ECLs), three intracellular loops, and a C-terminal tail. The structure of CCR5 was rendered by Pymol software based on a previously reported structure 4MBS ( 7 ). MPR and ECL2 are highlighted in blue and green, respectively. ECL2 contains a binding site for 2D7. (B) The amino acid sequence of N-terminal domain and first transmembrane helix (TM1) of CCR5. The antigenic regions were predicted by SVMTrip, PAP, and FBCPred methods and shown under the sequence of CCR5. (C) Balb/c mice were immunized with one dose of C17-keyhole limpet hemocyanin (KLH) plus complete Freund’s adjuvant, one dose of C17-KLH plus incomplete Freund’s adjuvant (IFA), and two doses of a mixture of C17-KLH, C17 peptide, and IFA. Sera were collected 1 week after the last immunization and antibody titers against C17-KLH, C17, or an irrelevant C34 peptide were measured by enzyme-linked immunosorbent assay. n = 3. (D) Total IgGs were purified from antiserum, designated as anti-C17. The binding of anti-C17 to CCR5 expressed on the cell surface of Ghost-R5 cells was determined by flow cytometry. Normal IgGs purified from the sera of un-immunized mice served as a negative control, while 2D7 served as a positive control. Alexa Fluor 647-conjugated anti-mouse IgG was used as the secondary antibody. C17 peptide and an irrelevant C34 peptide were used to confirm the binding specificity. Data are presented as mean ± SEM. All experiments were repeated twice with similar results.

    Techniques Used: Mouse Assay, Software, Binding Assay, Sequencing, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Purification, Flow Cytometry, Cytometry, Negative Control, Positive Control

    10) Product Images from "Interaction of a Specific Population of Human Embryonic Stem Cell-Derived Progenitor Cells with CD11b+ Cells Ameliorates Sepsis-Induced Lung Inflammatory Injury"

    Article Title: Interaction of a Specific Population of Human Embryonic Stem Cell-Derived Progenitor Cells with CD11b+ Cells Ameliorates Sepsis-Induced Lung Inflammatory Injury

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2010.09.041

    Transplantation of d7EB progenitor cells favorably alters cytokine production profile of CD11b+ cells in lungs. A: Reduced production of TNF-α and IFN-γ by CD11b+ cells obtained from lungs of mice that received d7EB cells. Lung CD11b+
    Figure Legend Snippet: Transplantation of d7EB progenitor cells favorably alters cytokine production profile of CD11b+ cells in lungs. A: Reduced production of TNF-α and IFN-γ by CD11b+ cells obtained from lungs of mice that received d7EB cells. Lung CD11b+

    Techniques Used: Transplantation Assay, Mouse Assay

    11) Product Images from "Dual-specificity Tyrosine Phosphorylation-regulated Kinase 1A (Dyrk1A) Modulates Serine/Arginine-rich Protein 55 (SRp55)-promoted Tau Exon 10 Inclusion *"

    Article Title: Dual-specificity Tyrosine Phosphorylation-regulated Kinase 1A (Dyrk1A) Modulates Serine/Arginine-rich Protein 55 (SRp55)-promoted Tau Exon 10 Inclusion *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.355412

    SRp55 interacts with Dyrk1A through RRM domain. A , co-immunoprecipitation of Dyrk1A by SRp55 required RRM. HA-tagged SRp55 FL or its deletion mutants and Dyrk1A were co-expressed in HEK-293FT cells for 48 h. The cell extracts were incubated with anti-HA prebound onto protein G beads. The bound proteins were subjected to Western blots using antibodies indicated under each blot. B , co-localization of SRp55 FL and its deletion mutants with Dyrk1A in the nucleus. HA-SRp55 and Dyrk1A were co-transfected into HeLa cells. After 48 h of transfection, the cells were fixed and immunostained by anti-HA or anti-Dyrk1A, followed by Cy3 anti-rabbit IgG or Alexa 488 anti-mouse IgG. IP , immunoprecipitation.
    Figure Legend Snippet: SRp55 interacts with Dyrk1A through RRM domain. A , co-immunoprecipitation of Dyrk1A by SRp55 required RRM. HA-tagged SRp55 FL or its deletion mutants and Dyrk1A were co-expressed in HEK-293FT cells for 48 h. The cell extracts were incubated with anti-HA prebound onto protein G beads. The bound proteins were subjected to Western blots using antibodies indicated under each blot. B , co-localization of SRp55 FL and its deletion mutants with Dyrk1A in the nucleus. HA-SRp55 and Dyrk1A were co-transfected into HeLa cells. After 48 h of transfection, the cells were fixed and immunostained by anti-HA or anti-Dyrk1A, followed by Cy3 anti-rabbit IgG or Alexa 488 anti-mouse IgG. IP , immunoprecipitation.

    Techniques Used: Immunoprecipitation, Incubation, Western Blot, Transfection

    Dyrk1A suppresses SRp55-promoted Tau exon 10 inclusion. A , effects of Dyrk1A or dominant negative Dyrk1A, Dyrk1A K188R , on SRp55-mediated Tau exon 10 splicing. Dyrk1A or Dyrk1A K188R was co-expressed with SRp55 in pCI/SI9-LI10 transfected HEK-293FT cells for 48 h. The alternative splicing products of Tau exon 10 were measured by RT-PCR. The ratio of inclusion and exclusion of Tau exon 10 is presented in the lower panel. B , syngeneic effect of Dyrk1A and siRNA of SRp55 ( siSRp55 ) on Tau exon 10 splicing. Dyrk1A or Dyrk1A K188R was co-expressed with siRNA of human SRp55 in pCI/SI9-LI10 transfected HEK-293FT cells for 48 h. The alternative splicing products of Tau exon were measured with RT-PCR. The ratio of inclusion and exclusion of Tau exon 10 is presented in the lower panel. C , mutation of SRp55 at Ser-303 to Ala enhanced its promotion in Tau exon 10 inclusion. Wild type SRp55 (SRp55 WT ) or its mutants (SRp55 S303A , SRp55 S316A , and SRp55 S280A ) were expressed in pCI/SI9-LI10-transfected HEK-293FT cells for 48 h. The alternative splicing products of Tau exon 10 were measured by RT-PCR. The ratio of inclusion and exclusion of Tau exon 10 is presented in the lower panel. D , Dyrk1A or Dyrk1A K188R differently affected the subcellular localization of SRp55. HA-SRp55 and Dyrk1A or Dyrk1A K188R were co-transfected into HeLa cells. After 48 h transfection, the cells were fixed and double-immunostained by anti-HA and anti-Dyrk1A, followed by Cy3 anti-rabbit IgG and Alexa 488 anti-mouse IgG. The results represent the means ± S.D. *, p
    Figure Legend Snippet: Dyrk1A suppresses SRp55-promoted Tau exon 10 inclusion. A , effects of Dyrk1A or dominant negative Dyrk1A, Dyrk1A K188R , on SRp55-mediated Tau exon 10 splicing. Dyrk1A or Dyrk1A K188R was co-expressed with SRp55 in pCI/SI9-LI10 transfected HEK-293FT cells for 48 h. The alternative splicing products of Tau exon 10 were measured by RT-PCR. The ratio of inclusion and exclusion of Tau exon 10 is presented in the lower panel. B , syngeneic effect of Dyrk1A and siRNA of SRp55 ( siSRp55 ) on Tau exon 10 splicing. Dyrk1A or Dyrk1A K188R was co-expressed with siRNA of human SRp55 in pCI/SI9-LI10 transfected HEK-293FT cells for 48 h. The alternative splicing products of Tau exon were measured with RT-PCR. The ratio of inclusion and exclusion of Tau exon 10 is presented in the lower panel. C , mutation of SRp55 at Ser-303 to Ala enhanced its promotion in Tau exon 10 inclusion. Wild type SRp55 (SRp55 WT ) or its mutants (SRp55 S303A , SRp55 S316A , and SRp55 S280A ) were expressed in pCI/SI9-LI10-transfected HEK-293FT cells for 48 h. The alternative splicing products of Tau exon 10 were measured by RT-PCR. The ratio of inclusion and exclusion of Tau exon 10 is presented in the lower panel. D , Dyrk1A or Dyrk1A K188R differently affected the subcellular localization of SRp55. HA-SRp55 and Dyrk1A or Dyrk1A K188R were co-transfected into HeLa cells. After 48 h transfection, the cells were fixed and double-immunostained by anti-HA and anti-Dyrk1A, followed by Cy3 anti-rabbit IgG and Alexa 488 anti-mouse IgG. The results represent the means ± S.D. *, p

    Techniques Used: Dominant Negative Mutation, Transfection, Reverse Transcription Polymerase Chain Reaction, Mutagenesis

    12) Product Images from "Single amino acid substitution in LC-CDR1 induces Russell body phenotype that attenuates cellular protein synthesis through eIF2α phosphorylation and thereby downregulates IgG secretion despite operational secretory pathway traffic"

    Article Title: Single amino acid substitution in LC-CDR1 induces Russell body phenotype that attenuates cellular protein synthesis through eIF2α phosphorylation and thereby downregulates IgG secretion despite operational secretory pathway traffic

    Journal: mAbs

    doi: 10.1080/19420862.2017.1314875

    Single amino acid substitution leads to a marked increase in Russell body phenotype occurrence during immunoglobulin biosynthesis. Fluorescent micrographs of HEK293 cells expressing the parental IgG or its N35W variant. On day-2 post transfection, HEK293 cells were resuspended in fresh cell culture media with or without 15 μg/ml BFA, then immediately seeded onto poly-lysine coated glass coverslips and statically cultured for 24 hr. On day-3, cells were fixed, permeabilized, and immuno-stained. Co-staining was performed by using FITC-conjugated anti-gamma chain and Texas Red-conjugated anti-kappa chain polyclonal antibodies. Green and red image fields were superimposed to create ‘merge’ views. DIC and ‘merge’ were superimposed to generate ‘overlay’ views. (A) Subcellular localization of gamma-chain and kappa-chain was visualized under steady-state normal cell growth conditions. Two representative image fields for the parental IgG expressing cells are shown in the first 2 rows. Two representative image fields for N35W variant IgG expressing cells are shown in rows 3 and 4. (B) Gamma- and kappa-chains of the parental IgG (first 2 rows) and N35W variant IgG (rows 3 to 5) were visualized after 24 hr BFA treatment. The RB phenotype frequency for each mAb under steady-state or after BFA treatment is stated in the text. Unlabeled scale bar represents 10 μm.
    Figure Legend Snippet: Single amino acid substitution leads to a marked increase in Russell body phenotype occurrence during immunoglobulin biosynthesis. Fluorescent micrographs of HEK293 cells expressing the parental IgG or its N35W variant. On day-2 post transfection, HEK293 cells were resuspended in fresh cell culture media with or without 15 μg/ml BFA, then immediately seeded onto poly-lysine coated glass coverslips and statically cultured for 24 hr. On day-3, cells were fixed, permeabilized, and immuno-stained. Co-staining was performed by using FITC-conjugated anti-gamma chain and Texas Red-conjugated anti-kappa chain polyclonal antibodies. Green and red image fields were superimposed to create ‘merge’ views. DIC and ‘merge’ were superimposed to generate ‘overlay’ views. (A) Subcellular localization of gamma-chain and kappa-chain was visualized under steady-state normal cell growth conditions. Two representative image fields for the parental IgG expressing cells are shown in the first 2 rows. Two representative image fields for N35W variant IgG expressing cells are shown in rows 3 and 4. (B) Gamma- and kappa-chains of the parental IgG (first 2 rows) and N35W variant IgG (rows 3 to 5) were visualized after 24 hr BFA treatment. The RB phenotype frequency for each mAb under steady-state or after BFA treatment is stated in the text. Unlabeled scale bar represents 10 μm.

    Techniques Used: Expressing, Variant Assay, Transfection, Cell Culture, Staining

    with red indicating high hydrophobicity and white indicating low hydrophobicity. The N35W variant has more exposed hydrophobic characteristics in the CDRs than does the parental mAb. LC-CDR1 (L1) and HC-CDR3 (H3) are long and are predicted to be highly dynamic in solution. The Trp residue of the N35W variant is likely to be solvent-exposed, presenting a hydrophobic patch that could render the antibody prone to non-specific interactions. (B, C, D) Cell culture media and whole cell lysate samples were prepared on day-7 post transfection and were subjected to SDS-PAGE followed by Western blot using rabbit anti-human IgG (H+L) polyclonal antibody. Samples in lanes 1–2 and lanes 3–5 were analyzed in the non-contiguous lanes of a single gel, but the intervening lanes were digitally removed to create the figures B–D. (B) A sample volume corresponding to the 5 μl of harvested cell culture medium was loaded per lane and analyzed under reducing conditions. Expected band for heavy chain (HC) and light chain (LC) subunits are marked by arrowhead. Determined secretion titers for this mAb production run are shown in lanes 1 and 2. (C) Whole cell lysates equivalent to 12,000–12,500 cells were loaded per lane under reducing conditions. Expected band for HC and LC subunits are marked by arrowhead. A faint band marked by asterisk in lane 5 is apparently a LC-N35W dimer species that was resistant to SDS treatment or disulfide reduction. Anti-GAPDH blot is shown at the bottom as a loading reference. (D) Cell culture media were analyzed under non-reducing conditions. LCs are secreted as a mixture of monomers (mono.) and covalent dimers (di.). Trimer and tetramer species are faintly detectable. (E) On day-2 post transfection, transfected cells were resuspended in fresh growth media with or without 15 μg/ml BFA and maintained in suspension format until day-3 when cell culture media and cell pellets were harvested and analyzed under reducing conditions. The amount of IgGs secreted to the culture medium during the 24 hr period is shown in the left panel. The amount of IgGs detected in the cell lysates is shown in the right panel. Anti-GAPDH blot is shown as a loading reference for cell lysate samples. (F, G) Cell viability and viable cell density were monitored daily by using automated cell counters. Data compiled from 6 independent experiments for the parental IgG and 4 independent runs for the N35W variant were used to calculate the average and standard deviation.
    Figure Legend Snippet: with red indicating high hydrophobicity and white indicating low hydrophobicity. The N35W variant has more exposed hydrophobic characteristics in the CDRs than does the parental mAb. LC-CDR1 (L1) and HC-CDR3 (H3) are long and are predicted to be highly dynamic in solution. The Trp residue of the N35W variant is likely to be solvent-exposed, presenting a hydrophobic patch that could render the antibody prone to non-specific interactions. (B, C, D) Cell culture media and whole cell lysate samples were prepared on day-7 post transfection and were subjected to SDS-PAGE followed by Western blot using rabbit anti-human IgG (H+L) polyclonal antibody. Samples in lanes 1–2 and lanes 3–5 were analyzed in the non-contiguous lanes of a single gel, but the intervening lanes were digitally removed to create the figures B–D. (B) A sample volume corresponding to the 5 μl of harvested cell culture medium was loaded per lane and analyzed under reducing conditions. Expected band for heavy chain (HC) and light chain (LC) subunits are marked by arrowhead. Determined secretion titers for this mAb production run are shown in lanes 1 and 2. (C) Whole cell lysates equivalent to 12,000–12,500 cells were loaded per lane under reducing conditions. Expected band for HC and LC subunits are marked by arrowhead. A faint band marked by asterisk in lane 5 is apparently a LC-N35W dimer species that was resistant to SDS treatment or disulfide reduction. Anti-GAPDH blot is shown at the bottom as a loading reference. (D) Cell culture media were analyzed under non-reducing conditions. LCs are secreted as a mixture of monomers (mono.) and covalent dimers (di.). Trimer and tetramer species are faintly detectable. (E) On day-2 post transfection, transfected cells were resuspended in fresh growth media with or without 15 μg/ml BFA and maintained in suspension format until day-3 when cell culture media and cell pellets were harvested and analyzed under reducing conditions. The amount of IgGs secreted to the culture medium during the 24 hr period is shown in the left panel. The amount of IgGs detected in the cell lysates is shown in the right panel. Anti-GAPDH blot is shown as a loading reference for cell lysate samples. (F, G) Cell viability and viable cell density were monitored daily by using automated cell counters. Data compiled from 6 independent experiments for the parental IgG and 4 independent runs for the N35W variant were used to calculate the average and standard deviation.

    Techniques Used: Variant Assay, Cell Culture, Transfection, SDS Page, Western Blot, Standard Deviation

    Transfected cells were first labeled for 30 min using Alexa Fluor 488 Click-iT® Plus OPP reagent to detect actively ongoing protein translation in situ (see Materials and Methods). The click-labeled cells were then immuno-stained with (A) Alexa Fluor 594-conjugated anti-human IgG (H+L) or (B) Texas Red-conjugated anti-gamma chain polyclonal antibodies. Green and red image fields were superimposed to create ‘merge’ views. DIC and green or DIC and red images were superimposed to generate ‘overlay’ views.
    Figure Legend Snippet: Transfected cells were first labeled for 30 min using Alexa Fluor 488 Click-iT® Plus OPP reagent to detect actively ongoing protein translation in situ (see Materials and Methods). The click-labeled cells were then immuno-stained with (A) Alexa Fluor 594-conjugated anti-human IgG (H+L) or (B) Texas Red-conjugated anti-gamma chain polyclonal antibodies. Green and red image fields were superimposed to create ‘merge’ views. DIC and green or DIC and red images were superimposed to generate ‘overlay’ views.

    Techniques Used: Transfection, Labeling, In Situ, Staining

    13) Product Images from "Norwalk Virus Does Not Replicate in Human Macrophages or Dendritic Cells Derived from the Peripheral Blood of Susceptible Humans"

    Article Title: Norwalk Virus Does Not Replicate in Human Macrophages or Dendritic Cells Derived from the Peripheral Blood of Susceptible Humans

    Journal: Virology

    doi: 10.1016/j.virol.2010.07.001

    NV protein expressing cells co-localize with some CX3CR1 + cells, but not with CD103 + cells by immunofluorescence
    Figure Legend Snippet: NV protein expressing cells co-localize with some CX3CR1 + cells, but not with CD103 + cells by immunofluorescence

    Techniques Used: Expressing, Immunofluorescence

    14) Product Images from "Myeloperoxidase-Derived Oxidants Induce Blood-Brain Barrier Dysfunction In Vitro and In Vivo"

    Article Title: Myeloperoxidase-Derived Oxidants Induce Blood-Brain Barrier Dysfunction In Vitro and In Vivo

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0064034

    Systemic inflammation induces cell-associated and extracellular (secreted) MPO. C57BL/6 mice received (A, D) PBS or (B, C, E, F) a single systemic LPS injection (250 µg LPS/30 g body weight; i.p.). After 6 h, animals were killed by cervical dislocation, brains were removed and snap frozen in liquid nitrogen. (A-C) Immunostaining of MPO was performed on sagittal cryosections (5 µm) of brain tissue using rabbit anti-human MPO IgG (1∶500) and HRP-labeled goat-anti rabbit IgG (1∶300) as primary and secondary antibodies. MPO (red) was visualized using the AEC system. Staining in a venule (B) and a smaller vessel (C) is shown. (D-F) Negative controls using rabbit non-immune IgG as primary antibody. Sections were counterstained with Mayer's hemalum. Arrows indicate cell-associated MPO, arrowheads indicate secreted MPO located at the abluminal side of vessels. Scale bars: 50 µm.
    Figure Legend Snippet: Systemic inflammation induces cell-associated and extracellular (secreted) MPO. C57BL/6 mice received (A, D) PBS or (B, C, E, F) a single systemic LPS injection (250 µg LPS/30 g body weight; i.p.). After 6 h, animals were killed by cervical dislocation, brains were removed and snap frozen in liquid nitrogen. (A-C) Immunostaining of MPO was performed on sagittal cryosections (5 µm) of brain tissue using rabbit anti-human MPO IgG (1∶500) and HRP-labeled goat-anti rabbit IgG (1∶300) as primary and secondary antibodies. MPO (red) was visualized using the AEC system. Staining in a venule (B) and a smaller vessel (C) is shown. (D-F) Negative controls using rabbit non-immune IgG as primary antibody. Sections were counterstained with Mayer's hemalum. Arrows indicate cell-associated MPO, arrowheads indicate secreted MPO located at the abluminal side of vessels. Scale bars: 50 µm.

    Techniques Used: Mouse Assay, Injection, Immunostaining, Labeling, Staining

    15) Product Images from "Mitochondrial-dependent Autoimmunity in Membranous Nephropathy of IgG4-related Disease"

    Article Title: Mitochondrial-dependent Autoimmunity in Membranous Nephropathy of IgG4-related Disease

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2015.03.003

    Patient's IgG4 recognized CAII on podocytes. (A) Western blotting of the 55 kDa recombinant human GST-tagged CAII (rhCAII, 0.5 μg) or human podocyte extracts (HPE, 20 μg) with patient's (first and second lanes) or control's serum (fourth lane). IgG-subclass specificity was evaluated using an anti-human IgG4-HRP antibody. The HPE membrane was stripped and reprobed with a specific anti-human CAII antibody (third lane). Molecular weights (kDa) shown on the left side. (B) Western blotting of purified CAII (pCAII, 0.5 μg) and the recombinant human GST-tagged SOD2 (rSOD2, 0.6 μg) with patient's serum. IgG-subclass specificity was evaluated using an anti-human IgG1-HRP antibody, anti-human IgG2-HRP antibody, anti-human IgG3-HRP antibody or anti-human IgG4-HRP antibody. (C) CAII expression evaluated using immunofluorescence analysis (left) or Western blotting (right) of podocyte crude plasma membranes (CPM, 20 μg). Scale bars 20 μm. (D) CAII immunogold staining in normal human kidney.
    Figure Legend Snippet: Patient's IgG4 recognized CAII on podocytes. (A) Western blotting of the 55 kDa recombinant human GST-tagged CAII (rhCAII, 0.5 μg) or human podocyte extracts (HPE, 20 μg) with patient's (first and second lanes) or control's serum (fourth lane). IgG-subclass specificity was evaluated using an anti-human IgG4-HRP antibody. The HPE membrane was stripped and reprobed with a specific anti-human CAII antibody (third lane). Molecular weights (kDa) shown on the left side. (B) Western blotting of purified CAII (pCAII, 0.5 μg) and the recombinant human GST-tagged SOD2 (rSOD2, 0.6 μg) with patient's serum. IgG-subclass specificity was evaluated using an anti-human IgG1-HRP antibody, anti-human IgG2-HRP antibody, anti-human IgG3-HRP antibody or anti-human IgG4-HRP antibody. (C) CAII expression evaluated using immunofluorescence analysis (left) or Western blotting (right) of podocyte crude plasma membranes (CPM, 20 μg). Scale bars 20 μm. (D) CAII immunogold staining in normal human kidney.

    Techniques Used: Western Blot, Recombinant, Purification, Expressing, Immunofluorescence, Staining

    Effect of IgG4 anti-CAII in sera of untreated IgG4-related disease patients. (A) Western blotting of purified CAII (pCAII, 0.6 μg). IgG-subclass specificity against CAII was evaluated using an anti-human IgG4-HRP antibody. (B) Intracellular pH detected using fluorimetric assay in podocytes exposed 6 h to IgG4 anti-CAII positive sera (CAII + sera), IgG4 anti-CAII negative serum (CAII − sera), control's serum (control's serum) or control medium (medium). °°p
    Figure Legend Snippet: Effect of IgG4 anti-CAII in sera of untreated IgG4-related disease patients. (A) Western blotting of purified CAII (pCAII, 0.6 μg). IgG-subclass specificity against CAII was evaluated using an anti-human IgG4-HRP antibody. (B) Intracellular pH detected using fluorimetric assay in podocytes exposed 6 h to IgG4 anti-CAII positive sera (CAII + sera), IgG4 anti-CAII negative serum (CAII − sera), control's serum (control's serum) or control medium (medium). °°p

    Techniques Used: Western Blot, Purification, Fluorimetry Assay

    16) Product Images from "A prophylactic α-Gal-based glycovaccine effectively protects against murine acute Chagas disease"

    Article Title: A prophylactic α-Gal-based glycovaccine effectively protects against murine acute Chagas disease

    Journal: NPJ Vaccines

    doi: 10.1038/s41541-019-0107-7

    Anti-Galα3LN-HSA Abs are α-Gal-specific, have lytic activity, and recognize parasite surface glycotopes. a Chemiluminescent enzyme-linked immunosorbent assay reactivity of sera from α1,3GalT-KO mice vaccinated with Galα3LN-HSA+/−LMPLA before and after green coffee bean α-galactosidase (α-Galase) treatment of the immobilized Galα3LN-HSA antigen. Purified human Chagas (Ch) anti-α-Gal immunoglobulin G (IgG) antibodies (Abs) and human serum pool from chronic Ch disease patients (ChHSP) were used as controls. RLU relative luminescence units. One-way ANOVA with Sidak’s multiple comparison test was performed comparing groups before (−) and after (+) α-galactosidase (α-Galase) treatment; p values indicate the significance of group before and after treatment. ** p
    Figure Legend Snippet: Anti-Galα3LN-HSA Abs are α-Gal-specific, have lytic activity, and recognize parasite surface glycotopes. a Chemiluminescent enzyme-linked immunosorbent assay reactivity of sera from α1,3GalT-KO mice vaccinated with Galα3LN-HSA+/−LMPLA before and after green coffee bean α-galactosidase (α-Galase) treatment of the immobilized Galα3LN-HSA antigen. Purified human Chagas (Ch) anti-α-Gal immunoglobulin G (IgG) antibodies (Abs) and human serum pool from chronic Ch disease patients (ChHSP) were used as controls. RLU relative luminescence units. One-way ANOVA with Sidak’s multiple comparison test was performed comparing groups before (−) and after (+) α-galactosidase (α-Galase) treatment; p values indicate the significance of group before and after treatment. ** p

    Techniques Used: Activity Assay, Chemiluminescent ELISA, Mouse Assay, Purification

    17) Product Images from "Humanization and Characterization of an Anti-Human TNF-? Murine Monoclonal Antibody"

    Article Title: Humanization and Characterization of an Anti-Human TNF-? Murine Monoclonal Antibody

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0016373

    Binding affinities of h357 IgG to soluble and membrane form TNF-α. (A) Binding affinities of different TNF-α anatagonists to soluble TNF-α. Microtiter plates with the anti-human IgG Fcγ fragment captured h357 (•), etanercept (▾) and adalimumab (△) and the anti-mouse IgG Fcγ fragment captured m357 (○) were incubated with various concentrations of soluble TNF-α for 1 hr at 37°C. After washing, the bound TNF-α was detected by a mouse monoclonal anti-TNF-α antibody conjugated with horseradish peroxidase. Absorbance was read at 450 nm on a microplate reader. (B) The saturation binding curves of m357 and h357 IgGs to transmembrane TNF-α. Transmembrane TNF-α-transfected NS0 cells were incubated with serial log dilutions of m357 (○) or h357 (•) antibody for 1 hr at 4°C. Cell were washed and incubated with Alexa Fluor 488 goat anti-mouse IgG (H+L) for m357 and Alexa Fluor 647 goat anti-human IgG (H+L) for h357, respectively for 1 hr at 4°C. Cells were then washed and analyzed by FACSCalibur flow cytometer.
    Figure Legend Snippet: Binding affinities of h357 IgG to soluble and membrane form TNF-α. (A) Binding affinities of different TNF-α anatagonists to soluble TNF-α. Microtiter plates with the anti-human IgG Fcγ fragment captured h357 (•), etanercept (▾) and adalimumab (△) and the anti-mouse IgG Fcγ fragment captured m357 (○) were incubated with various concentrations of soluble TNF-α for 1 hr at 37°C. After washing, the bound TNF-α was detected by a mouse monoclonal anti-TNF-α antibody conjugated with horseradish peroxidase. Absorbance was read at 450 nm on a microplate reader. (B) The saturation binding curves of m357 and h357 IgGs to transmembrane TNF-α. Transmembrane TNF-α-transfected NS0 cells were incubated with serial log dilutions of m357 (○) or h357 (•) antibody for 1 hr at 4°C. Cell were washed and incubated with Alexa Fluor 488 goat anti-mouse IgG (H+L) for m357 and Alexa Fluor 647 goat anti-human IgG (H+L) for h357, respectively for 1 hr at 4°C. Cells were then washed and analyzed by FACSCalibur flow cytometer.

    Techniques Used: Binding Assay, Incubation, Transfection, Flow Cytometry, Cytometry

    18) Product Images from "Role of Fibronectin in the Adhesion of Acinetobacter baumannii to Host Cells"

    Article Title: Role of Fibronectin in the Adhesion of Acinetobacter baumannii to Host Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0033073

    Involvement of fibronectin in the adherence of A. baumannii to human lung epithelial cells. A549 cells were pretreated with RGD (0.5 and 5 mg/mL) ( A ), rabbit anti-human fibronectin (1∶25), rat anti-human E-cadherin (1∶25) or control mouse IgG (1∶25) ( B ) and infected with 10 8 cfu/mL of A. baumannii ATCC 19606, 77 or 113-16 strain for 2 h. Adherence assay was performed as described in materials and methods . The effect of treatment on A. baumannii adherence to A549 cells is expressed as the percentage of total untreated A. baumannii adhered to A549 cells. Immunostaining for A549 cells fibronectin and A. baumannii OMPs in infected A549 cells with 10 8 cfu/mL A. baumannii ATCC 19606, 77 or 113 strains for 2 h ( C ) and 24 h ( D ) were performed and imaged by immunofluorescence microscopy. Fibronectin of A549 cells and OMPs of A. baumannii strains were detected by rabbit anti-human fibronectin and mouse anti- A. baumannii OMPs antibodies and labeled with Alexa594 and Alexa488-tagged the secondary antibodies that appeared red and green, respectively. Blue staining shows the location of A549 cells nucleus. Representative results of three independent experiments are shown and data are the means ± SEM. P
    Figure Legend Snippet: Involvement of fibronectin in the adherence of A. baumannii to human lung epithelial cells. A549 cells were pretreated with RGD (0.5 and 5 mg/mL) ( A ), rabbit anti-human fibronectin (1∶25), rat anti-human E-cadherin (1∶25) or control mouse IgG (1∶25) ( B ) and infected with 10 8 cfu/mL of A. baumannii ATCC 19606, 77 or 113-16 strain for 2 h. Adherence assay was performed as described in materials and methods . The effect of treatment on A. baumannii adherence to A549 cells is expressed as the percentage of total untreated A. baumannii adhered to A549 cells. Immunostaining for A549 cells fibronectin and A. baumannii OMPs in infected A549 cells with 10 8 cfu/mL A. baumannii ATCC 19606, 77 or 113 strains for 2 h ( C ) and 24 h ( D ) were performed and imaged by immunofluorescence microscopy. Fibronectin of A549 cells and OMPs of A. baumannii strains were detected by rabbit anti-human fibronectin and mouse anti- A. baumannii OMPs antibodies and labeled with Alexa594 and Alexa488-tagged the secondary antibodies that appeared red and green, respectively. Blue staining shows the location of A549 cells nucleus. Representative results of three independent experiments are shown and data are the means ± SEM. P

    Techniques Used: Infection, Immunostaining, Immunofluorescence, Microscopy, Labeling, Staining

    19) Product Images from "Secreted NS1 of Dengue Virus Attaches to the Surface of Cells via Interactions with Heparan Sulfate and Chondroitin Sulfate E"

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

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.0030183

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

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

    20) Product Images from "Histopathologic Overlap between Fibrosing Mediastinitis and IgG4-Related Disease"

    Article Title: Histopathologic Overlap between Fibrosing Mediastinitis and IgG4-Related Disease

    Journal: International Journal of Rheumatology

    doi: 10.1155/2012/207056

    Histopathological changes and IgG4 immunostaining demonstrate an overlap between FM and IgG4-RD in a subset of patients ( n = 3), (a). cell rich, storiform fibrosis, (b). vascular inflammation and (c). IgG4 positive plasma cells. (representative images selected from 3 patients).
    Figure Legend Snippet: Histopathological changes and IgG4 immunostaining demonstrate an overlap between FM and IgG4-RD in a subset of patients ( n = 3), (a). cell rich, storiform fibrosis, (b). vascular inflammation and (c). IgG4 positive plasma cells. (representative images selected from 3 patients).

    Techniques Used: Immunostaining

    Number of IgG4-positive plasma cells and the corresponding IgG4 + /IgG + ratios in FM patients ( n = 15, 3 patients had no IgG4 positive plasma cells).
    Figure Legend Snippet: Number of IgG4-positive plasma cells and the corresponding IgG4 + /IgG + ratios in FM patients ( n = 15, 3 patients had no IgG4 positive plasma cells).

    Techniques Used:

    21) Product Images from "IgG1 antibodies to acetylcholine receptors in 'seronegative' myasthenia gravis †"

    Article Title: IgG1 antibodies to acetylcholine receptors in 'seronegative' myasthenia gravis †

    Journal: Brain

    doi: 10.1093/brain/awn092

    Thymic changes involved in AChR-MGlow and SNMG patients. ( A ) Size of infiltrates as a percentage of thymic tissue in all MG and control groups. ( B and C ) Distribution of myoid cells (stained for desmin, red) around or within the infiltrates/germinal centres (stained for CD20 on B cells, green) in AChR-MGlow ( B ) and in SNMG ( C ) thymi, and quantified in ( D ). ( E and F ) Signs of complement activation (deposits of C3b, red) on myoid cells (green) in AChR-MGlow ( E ) and SNMG thymus ( F ). ( G ) Correlation between the score of IgG binding to AChR clusters and the percentage of C3b positive myoid cells exposed to the infiltrates. In ( A ) and ( D ), column and symbols represents median and range, and lines show median values. In ( B ) and ( C ), bar = 100 μm. Data in ( A ) and ( D ) for MG patients, apart from AChR-MGlow patients, are derived from Leite et al ., 2005 , 2007 . Data for AChR-MGlow are previously unpublished.
    Figure Legend Snippet: Thymic changes involved in AChR-MGlow and SNMG patients. ( A ) Size of infiltrates as a percentage of thymic tissue in all MG and control groups. ( B and C ) Distribution of myoid cells (stained for desmin, red) around or within the infiltrates/germinal centres (stained for CD20 on B cells, green) in AChR-MGlow ( B ) and in SNMG ( C ) thymi, and quantified in ( D ). ( E and F ) Signs of complement activation (deposits of C3b, red) on myoid cells (green) in AChR-MGlow ( E ) and SNMG thymus ( F ). ( G ) Correlation between the score of IgG binding to AChR clusters and the percentage of C3b positive myoid cells exposed to the infiltrates. In ( A ) and ( D ), column and symbols represents median and range, and lines show median values. In ( B ) and ( C ), bar = 100 μm. Data in ( A ) and ( D ) for MG patients, apart from AChR-MGlow patients, are derived from Leite et al ., 2005 , 2007 . Data for AChR-MGlow are previously unpublished.

    Techniques Used: Staining, Activation Assay, Binding Assay, Derivative Assay

    AChR clustering enhances detection of AChR antibodies. ( A ) Binding of IgG antibodies (red) to adult AChR clustered by rapsyn-EGFP on HEK cell surface (green) with scores shown on the right. ( B ) Scores for all samples tested. Median values are shown, and number of sera tested in brackets. ( C ) Comparison between IgG binding scores to unclustered and clustered AChR for all sera tested. Columns show means +SEM. There were differences between binding to clustered and unclustered AChR for AChR-MGhigh, AChR-MGlow and SNMG sera (all P
    Figure Legend Snippet: AChR clustering enhances detection of AChR antibodies. ( A ) Binding of IgG antibodies (red) to adult AChR clustered by rapsyn-EGFP on HEK cell surface (green) with scores shown on the right. ( B ) Scores for all samples tested. Median values are shown, and number of sera tested in brackets. ( C ) Comparison between IgG binding scores to unclustered and clustered AChR for all sera tested. Columns show means +SEM. There were differences between binding to clustered and unclustered AChR for AChR-MGhigh, AChR-MGlow and SNMG sera (all P

    Techniques Used: Binding Assay

    ( A ) FACS analysis of binding demonstrating the gating, based on untreated cells, and showing some example profiles. ( B ) Scatter plots of FACS analyses, comparing IgG binding (percentage of gated) to cells expressing unclustered or clustered AChR. The cut-off was based on the mean +2 SDs of results from seven healthy controls binding to clustered AChR. ( C ) Correlation between positive cells (percentage of gated) on FACS and binding scores obtained by visual inspection for the 39 sera tested.
    Figure Legend Snippet: ( A ) FACS analysis of binding demonstrating the gating, based on untreated cells, and showing some example profiles. ( B ) Scatter plots of FACS analyses, comparing IgG binding (percentage of gated) to cells expressing unclustered or clustered AChR. The cut-off was based on the mean +2 SDs of results from seven healthy controls binding to clustered AChR. ( C ) Correlation between positive cells (percentage of gated) on FACS and binding scores obtained by visual inspection for the 39 sera tested.

    Techniques Used: FACS, Binding Assay, Expressing

    Characterization of IgG antibodies to clustered adult AChR in AChR-MGlow and in SNMG samples. ( A ) Merged images show predominantly IgG1 (orange), and little IgG4 AChR antibody. ( B ) In the presence of fresh human serum and anti-human C3b or anti-human C5-9 (membrane attack complex, MAC), activated complement deposits were found in both AChR-MG and SNMG samples. Shown are C3b deposits for one AChR-MG serum, and C3b or MAC deposits for three SNMG sera. ( C ) Scores of IgG1 and IgG4 binding, and detection of C3b and MAC deposits in nine SNMG samples tested. Lines show median values and number of samples tested are in brackets. Sera from healthy individuals did not show any evidence of complement deposition.
    Figure Legend Snippet: Characterization of IgG antibodies to clustered adult AChR in AChR-MGlow and in SNMG samples. ( A ) Merged images show predominantly IgG1 (orange), and little IgG4 AChR antibody. ( B ) In the presence of fresh human serum and anti-human C3b or anti-human C5-9 (membrane attack complex, MAC), activated complement deposits were found in both AChR-MG and SNMG samples. Shown are C3b deposits for one AChR-MG serum, and C3b or MAC deposits for three SNMG sera. ( C ) Scores of IgG1 and IgG4 binding, and detection of C3b and MAC deposits in nine SNMG samples tested. Lines show median values and number of samples tested are in brackets. Sera from healthy individuals did not show any evidence of complement deposition.

    Techniques Used: Binding Assay

    Detection of MuSK-MG antibodies binding to MuSK-EGFP transfected HEK cells and their characterization. ( A ) Scores of IgG binding to MuSK of all serum samples. ( B ) Merged pictures of IgG1 and IgG4 binding (yellow/orange) showing predominantly IgG4 antibodies but some IgG1. ( C ) C3b deposition (yellow/orange) in the presence of fresh human serum. ( D ) Scores of IgM, IgG1 and IgG4 antibody binding and scores of C3b deposition. Lines show median values and number of samples tested are in brackets.
    Figure Legend Snippet: Detection of MuSK-MG antibodies binding to MuSK-EGFP transfected HEK cells and their characterization. ( A ) Scores of IgG binding to MuSK of all serum samples. ( B ) Merged pictures of IgG1 and IgG4 binding (yellow/orange) showing predominantly IgG4 antibodies but some IgG1. ( C ) C3b deposition (yellow/orange) in the presence of fresh human serum. ( D ) Scores of IgM, IgG1 and IgG4 antibody binding and scores of C3b deposition. Lines show median values and number of samples tested are in brackets.

    Techniques Used: Binding Assay, Transfection

    22) Product Images from "Optimization of incubation conditions of Plasmodium falciparum antibody multiplex assays to measure IgG, IgG1–4, IgM and IgE using standard and customized reference pools for sero-epidemiological and vaccine studies"

    Article Title: Optimization of incubation conditions of Plasmodium falciparum antibody multiplex assays to measure IgG, IgG1–4, IgM and IgE using standard and customized reference pools for sero-epidemiological and vaccine studies

    Journal: Malaria Journal

    doi: 10.1186/s12936-018-2369-3

    RTS,S-specific responses measured in the WHO reference reagent, IgM pool and samples from RTS,S-vaccinated children. The 3 samples from RTS,S vaccinated children were of high, medium and low CSP IgG titres. a – g IgG, IgG 1–4 , IgM and IgE levels to RTS,S-specific antigens measured in the WHO reference reagent; IgG, IgG 1, IgG2 and IgG4 also measured in RTS,S-vaccinated children; h IgM levels to RTS,S-specific antigens measured in the IgM pool vs. RTS,S-vaccinated children. The plots represent the levels of antibodies measured in serial dilutions of the positive pools (1:3 starting at 1:50 for IgG, IgG 1–4 and IgM; and 1:2 starting at 1:10 for IgE), and the RTS,S vaccinees samples (1:10 starting at 1:500 for IgG, 1:100 for IgM, 1:50 for IgG 1–4 ; and 1:2 starting at 1:10 for IgE). Isolated dots represent the levels measured in the technical blanks
    Figure Legend Snippet: RTS,S-specific responses measured in the WHO reference reagent, IgM pool and samples from RTS,S-vaccinated children. The 3 samples from RTS,S vaccinated children were of high, medium and low CSP IgG titres. a – g IgG, IgG 1–4 , IgM and IgE levels to RTS,S-specific antigens measured in the WHO reference reagent; IgG, IgG 1, IgG2 and IgG4 also measured in RTS,S-vaccinated children; h IgM levels to RTS,S-specific antigens measured in the IgM pool vs. RTS,S-vaccinated children. The plots represent the levels of antibodies measured in serial dilutions of the positive pools (1:3 starting at 1:50 for IgG, IgG 1–4 and IgM; and 1:2 starting at 1:10 for IgE), and the RTS,S vaccinees samples (1:10 starting at 1:500 for IgG, 1:100 for IgM, 1:50 for IgG 1–4 ; and 1:2 starting at 1:10 for IgE). Isolated dots represent the levels measured in the technical blanks

    Techniques Used: Isolation

    23) Product Images from "Lateral Flow Test Using Echinococcus granulosus Native Antigen B and Comparison of IgG and IgG4 Dipsticks for Detection of Human Cystic Echinococcosis"

    Article Title: Lateral Flow Test Using Echinococcus granulosus Native Antigen B and Comparison of IgG and IgG4 Dipsticks for Detection of Human Cystic Echinococcosis

    Journal: The American Journal of Tropical Medicine and Hygiene

    doi: 10.4269/ajtmh.14-0170

    Representative native AgB dipsticks probed with colloidal gold-conjugated anti-human IgG. Lanes 1–3 show positive test results with serum samples from individual CE patients. Lane 4 shows a negative test result with a serum sample from a healthy
    Figure Legend Snippet: Representative native AgB dipsticks probed with colloidal gold-conjugated anti-human IgG. Lanes 1–3 show positive test results with serum samples from individual CE patients. Lane 4 shows a negative test result with a serum sample from a healthy

    Techniques Used:

    Representative AgB dipsticks probed with colloidal gold-conjugated anti-human IgG4. Lanes 1–3 show positive test results with serum samples from individual CE patients. Lane 4 shows a negative test result with a serum sample from a healthy individual.
    Figure Legend Snippet: Representative AgB dipsticks probed with colloidal gold-conjugated anti-human IgG4. Lanes 1–3 show positive test results with serum samples from individual CE patients. Lane 4 shows a negative test result with a serum sample from a healthy individual.

    Techniques Used:

    24) Product Images from "Complement inhibition by hydroxychloroquine prevents placental and fetal brain abnormalities in antiphospholipid syndrome"

    Article Title: Complement inhibition by hydroxychloroquine prevents placental and fetal brain abnormalities in antiphospholipid syndrome

    Journal: Journal of autoimmunity

    doi: 10.1016/j.jaut.2016.04.008

    Immunohistochemical studies to detect deposition of aβ2GPI and NHIgG in placenta and fetal brain. Expression of β 2 GPI protein in placenta and fetal brain in control mice . aβ2GPI and NHIgG deposition in mouse tissue (placenta and fetal cortical brain) was detected using an antihuman IgG antibody labelled with Texas Red. A- detection of aβ 2 GPI in the placenta from mouse injected aβ 2 GPI. B- Detection of aβ 2 GPI in fetal cortical brain from a mouse injected with aβ 2 GPI. C- Detection of NHIgG in the placenta from a mouse injected with NHIgG. D- Detection of NHIgG in the fetal cortical brain in a mouse injected with NHIgG. Expression of β 2 GPI in placenta (E) and fetal brain (F) was detected with FITC-conjugated antibodies (green fluorescence). Diamidino-2-phenylindole (DAPI) was used for nuclear counterstains in all immunofluorescence studies. Data are representative of observations in 5–6 mice per group. 10 views per slide were analyzed in each experimental condition.
    Figure Legend Snippet: Immunohistochemical studies to detect deposition of aβ2GPI and NHIgG in placenta and fetal brain. Expression of β 2 GPI protein in placenta and fetal brain in control mice . aβ2GPI and NHIgG deposition in mouse tissue (placenta and fetal cortical brain) was detected using an antihuman IgG antibody labelled with Texas Red. A- detection of aβ 2 GPI in the placenta from mouse injected aβ 2 GPI. B- Detection of aβ 2 GPI in fetal cortical brain from a mouse injected with aβ 2 GPI. C- Detection of NHIgG in the placenta from a mouse injected with NHIgG. D- Detection of NHIgG in the fetal cortical brain in a mouse injected with NHIgG. Expression of β 2 GPI in placenta (E) and fetal brain (F) was detected with FITC-conjugated antibodies (green fluorescence). Diamidino-2-phenylindole (DAPI) was used for nuclear counterstains in all immunofluorescence studies. Data are representative of observations in 5–6 mice per group. 10 views per slide were analyzed in each experimental condition.

    Techniques Used: Immunohistochemistry, Expressing, Mouse Assay, Injection, Fluorescence, Immunofluorescence

    25) Product Images from "?2-Glycoprotein I-dependent and -independent anticardiolipin antibody in non-obese diabetic (NOD) mice"

    Article Title: ?2-Glycoprotein I-dependent and -independent anticardiolipin antibody in non-obese diabetic (NOD) mice

    Journal: Clinical and Experimental Immunology

    doi: 10.1046/j.1365-2249.1998.00476.x

    Inhibition test and IgG subclass distribution of β 2 -glycoprotein I (β 2 -GPI)-dependent and -independent anticardiolipin antibodies (aCL). (a) Inhibition of autoantibody to β 2 -GPI by the fluid-phase complex of β 2 -GPI and CL. The β 2 -GPI-dependent aCL-positive serum was preincubated with varying amounts of either β 2 -GPI in the presence or absence of CL: β 2 -GPI (○), β 2 -GPI + CL (•). (c) Inhibition of autoantibody to CL by CL or β 2 -GPI. The β 2 -GPI-independent aCL-positive serum was preincubated with varying amounts of either CL or β 2 -GPI: CL (•), β 2 -GPI (○). (b,d) IgG subclass distribution of β 2 -GPI-dependent aCL (b) and β 2 -GPI-independent aCL (d) in the NOD mice sera by an ELISA using mouse monoclonal IgG subclass-specific antibodies. Each point indicates the absorbance of an individual serum.
    Figure Legend Snippet: Inhibition test and IgG subclass distribution of β 2 -glycoprotein I (β 2 -GPI)-dependent and -independent anticardiolipin antibodies (aCL). (a) Inhibition of autoantibody to β 2 -GPI by the fluid-phase complex of β 2 -GPI and CL. The β 2 -GPI-dependent aCL-positive serum was preincubated with varying amounts of either β 2 -GPI in the presence or absence of CL: β 2 -GPI (○), β 2 -GPI + CL (•). (c) Inhibition of autoantibody to CL by CL or β 2 -GPI. The β 2 -GPI-independent aCL-positive serum was preincubated with varying amounts of either CL or β 2 -GPI: CL (•), β 2 -GPI (○). (b,d) IgG subclass distribution of β 2 -GPI-dependent aCL (b) and β 2 -GPI-independent aCL (d) in the NOD mice sera by an ELISA using mouse monoclonal IgG subclass-specific antibodies. Each point indicates the absorbance of an individual serum.

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

    Immunoperoxidase staining of cryostat pancreas sections of a 20-week-old NOD female mouse using different MoAbs defining IgG1 (A), IgG2a (B), IgG2b (C) and IgG3 (D)-positive B lymphocytes.
    Figure Legend Snippet: Immunoperoxidase staining of cryostat pancreas sections of a 20-week-old NOD female mouse using different MoAbs defining IgG1 (A), IgG2a (B), IgG2b (C) and IgG3 (D)-positive B lymphocytes.

    Techniques Used: Immunoperoxidase Staining

    26) Product Images from "Placental Malaria Induces Variant-Specific Antibodies of the Cytophilic Subtypes Immunoglobulin G1 (IgG1) and IgG3 That Correlate with Adhesion Inhibitory Activity "

    Article Title: Placental Malaria Induces Variant-Specific Antibodies of the Cytophilic Subtypes Immunoglobulin G1 (IgG1) and IgG3 That Correlate with Adhesion Inhibitory Activity

    Journal: Infection and Immunity

    doi: 10.1128/IAI.73.9.5903-5907.2005

    Associations between binding of IgM and IgG subtypes to VSA expressed by the P. falciparum line CS2 and the ability of serum or plasma to inhibit adhesion of CS2-infected erythrocytes to chondroitin sulfate A. Levels of antibodies in serum or plasma from
    Figure Legend Snippet: Associations between binding of IgM and IgG subtypes to VSA expressed by the P. falciparum line CS2 and the ability of serum or plasma to inhibit adhesion of CS2-infected erythrocytes to chondroitin sulfate A. Levels of antibodies in serum or plasma from

    Techniques Used: Binding Assay, Infection

    Levels of IgG subtypes and IgM binding to VSA expressed by the chondroitin sulfate A-adherent P. falciparum line CS2. Flow cytometry was used to measure antibodies to CS2-VSA in serum/plasma from PG and MG women with histologically confirmed placental
    Figure Legend Snippet: Levels of IgG subtypes and IgM binding to VSA expressed by the chondroitin sulfate A-adherent P. falciparum line CS2. Flow cytometry was used to measure antibodies to CS2-VSA in serum/plasma from PG and MG women with histologically confirmed placental

    Techniques Used: Binding Assay, Flow Cytometry, Cytometry

    27) Product Images from "Complement-Related Proteins Control the Flavivirus Infection of Aedes aegypti by Inducing Antimicrobial Peptides"

    Article Title: Complement-Related Proteins Control the Flavivirus Infection of Aedes aegypti by Inducing Antimicrobial Peptides

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004027

    A Scavenger receptor-C with CCP domains recognizes DENV-2 in vitro and in vivo . (A) Percentage of amino acid identity of insect SR-Cs. (B) Schematic representation of SR-Cs in A. aegypti and D. melanogaster . The functional modules were predicted in SMART ( http://smart.embl-heidelberg.de/smart/set_mode.cgi?GENOMIC=1 ) and Pfam websites ( http://pfam.sanger.ac.uk/ ). (C) Expression and purification of AaSR-C from Drosophila S2 cells. The extracellular region (AaSR-C-Ex) or full length (AaSR-C-Full) AaSR-C was cloned into the pMT/BiP/V5-His-A expression vector. The recombinant plasmids were transfected into Drosophila S2 cells, and their expression was probed using an anti-V5 mAb. The supernatant or lysates from mock-transfected S2 cells was used as the mock control (Left panel). Recombinant AaSR-C-Ex, produced in Drosophila cells, was purified using an Ni-His column (Right panel). (D) AaSR-C-Ex interacted with DENV-2 E proteins in co-immunoprecipitation assay. Purified AaSR-C-Ex (V5) and DENV-2 E (FLAG) were used to investigate the interaction of the proteins. Control rabbit IgG was used as a mock control to exclude non-specific interactions. The protein complex was pulled down with a rabbit anti-FLAG antibody and detected using a mouse anti-V5 antibody. We reproduced the experiment 3 times. (E) AaSR-C-Ex captured DENV-2 virions in an ELISA. Binding was probed using the flavivirus E mAb 4G2. The data are presented as the mean ± standard error. The experiment was reproduced 3 times. (F) AaSR-C bound DENV-2 virions on the cell surface. A Cu 2+ -inducible stable S2 cell line was generated to express the full-length AaSR-C. DENV-2 virions were incubated with AaSR-C expressing cells at 4°C for 1 hr. Non-induced stable cells and empty vector-transfected S2 cells containing virions served as the mock control groups. The interaction between AaSR-C and DENV was measured through flow cytometry. The DENV virions were stained using the flaviviral E mAb 4G2 and anti-mouse IgG Alexa-488; AaSR-C was probed using a Myc mAb and anti-rabbit IgG Phycoerythrin (PE). The data was analyzed using FlowJo software. The presented data was representative of 3 independent experiments with similar results. (G) The in vivo association between AaSR-C and DENV-2 in A.aegypti hemocytes. Hemolymph was collected from uninfected mosquitoes, AaSR-C silenced infected mosquitoes and GFP dsRNA treated infected mosquitoes to undergo immunofluorescence staining. AaSR-C was stained with anti-rabbit IgG Alexa-488 (Green), and the DENV-2 E protein was identified using anti-mouse IgG Alexa-546 (Red). Nuclei were stained blue with To-Pro-3 iodide (Blue). Images were examined using a Zeiss LSM 780 meta confocal 63×objective lens.
    Figure Legend Snippet: A Scavenger receptor-C with CCP domains recognizes DENV-2 in vitro and in vivo . (A) Percentage of amino acid identity of insect SR-Cs. (B) Schematic representation of SR-Cs in A. aegypti and D. melanogaster . The functional modules were predicted in SMART ( http://smart.embl-heidelberg.de/smart/set_mode.cgi?GENOMIC=1 ) and Pfam websites ( http://pfam.sanger.ac.uk/ ). (C) Expression and purification of AaSR-C from Drosophila S2 cells. The extracellular region (AaSR-C-Ex) or full length (AaSR-C-Full) AaSR-C was cloned into the pMT/BiP/V5-His-A expression vector. The recombinant plasmids were transfected into Drosophila S2 cells, and their expression was probed using an anti-V5 mAb. The supernatant or lysates from mock-transfected S2 cells was used as the mock control (Left panel). Recombinant AaSR-C-Ex, produced in Drosophila cells, was purified using an Ni-His column (Right panel). (D) AaSR-C-Ex interacted with DENV-2 E proteins in co-immunoprecipitation assay. Purified AaSR-C-Ex (V5) and DENV-2 E (FLAG) were used to investigate the interaction of the proteins. Control rabbit IgG was used as a mock control to exclude non-specific interactions. The protein complex was pulled down with a rabbit anti-FLAG antibody and detected using a mouse anti-V5 antibody. We reproduced the experiment 3 times. (E) AaSR-C-Ex captured DENV-2 virions in an ELISA. Binding was probed using the flavivirus E mAb 4G2. The data are presented as the mean ± standard error. The experiment was reproduced 3 times. (F) AaSR-C bound DENV-2 virions on the cell surface. A Cu 2+ -inducible stable S2 cell line was generated to express the full-length AaSR-C. DENV-2 virions were incubated with AaSR-C expressing cells at 4°C for 1 hr. Non-induced stable cells and empty vector-transfected S2 cells containing virions served as the mock control groups. The interaction between AaSR-C and DENV was measured through flow cytometry. The DENV virions were stained using the flaviviral E mAb 4G2 and anti-mouse IgG Alexa-488; AaSR-C was probed using a Myc mAb and anti-rabbit IgG Phycoerythrin (PE). The data was analyzed using FlowJo software. The presented data was representative of 3 independent experiments with similar results. (G) The in vivo association between AaSR-C and DENV-2 in A.aegypti hemocytes. Hemolymph was collected from uninfected mosquitoes, AaSR-C silenced infected mosquitoes and GFP dsRNA treated infected mosquitoes to undergo immunofluorescence staining. AaSR-C was stained with anti-rabbit IgG Alexa-488 (Green), and the DENV-2 E protein was identified using anti-mouse IgG Alexa-546 (Red). Nuclei were stained blue with To-Pro-3 iodide (Blue). Images were examined using a Zeiss LSM 780 meta confocal 63×objective lens.

    Techniques Used: In Vitro, In Vivo, Functional Assay, Expressing, Purification, Clone Assay, Plasmid Preparation, Recombinant, Transfection, Produced, Co-Immunoprecipitation Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Generated, Incubation, Flow Cytometry, Cytometry, Staining, Software, Infection, Immunofluorescence

    AaMCR and AaSR-C function in a pathway that opposes DENV-2 infection. (A) The interaction between 3 AaMCR fragments and AaSR-C in co-IP assays. Three AaMCR gene fragments were cloned into the pMT/BiP/V5-His A vector. The recombinant plasmids were transiently transfected into S2 cells. The cell supernatant was used for investigation of the AaMCR/AaSR-C interaction. The protein complex was pulled down with a rabbit anti-V5 antibody and probed using a mouse anti-HA antibody. The experiment was reproduced 3 times. (B) Expression and purification of AaMCR-a in Drosophila S2 cells. The purified AaMCR-a was separated through SDS-PAGE (Left Panel) and detected with an anti-HA antibody via western blotting (Right Panel). The supernatant from empty vector-transfected S2 cells was used as the mock control. (C) AaSR-C-Ex acted as an adaptor in the interaction between the AaMCR-a and DENV-2 E proteins. The purified AaSR-C-Ex, AaMCR-a and DENV-2 E proteins were mixed and pulled down with a mouse anti-HA antibody (AaMCR-a) and detected using a rabbit anti-V5 antibody (AaSR-C) and anti-FLAG-HRP antibody (DENV-2 E). The experiment was repeated 3 times with similar results. (D) AaSR-C-Ex connected AaMCR-a to DENV-2 virions. Purified AaMCR-a or BSA was pre-coated into the ELISA plate wells. DENV-2 virions either mixed with AaSR-C-Ex or without AaSR-C-Ex were added to the protein-coated wells. The signal was detected using the flavivirus E mAb 4G2. The data are expressed as the mean ± standard error. The experiment was reproduced by 3 times with similar results. (E) Double knockdown of AaMCR and AaSR-C showed similar effects on DENV-2 infection to individual knockdown. Both AaSR-C (i) and AaMCR (ii) were knocked down using a dsRNA mixture in the AaSR-C / AaMCR co-silenced group. DENV-2 replication and the numbers of infectious DENV-2 virions in the mosquitoes were measured via qPCR (iii) and plaque assays (iv). Statistical analysis was performed using the non-parametric Mann-Whitney test. The data on gene silencing (i, ii) and from plaque assays (iv) are expressed as the mean ± standard error. The horizontal line depicts the median (iii). Each dot represents an individual mosquito. The result was representative of 3 independent experiments. (F) Immunostaining of AaMCR, AaSR-C and DENV-2 in A. aegypti hemocytes. The hemocytes were dissected from uninfected mosquitoes, AaMCR and/or AaSR-C silenced infected mosquitoes and GFP dsRNA treated infected mosquitoes at 6 days post-infection. AaSR-C was stained with anti-rabbit IgG Alexa-488 (Green); AaMCR was probed using anti-mouse IgG Alexa-546 (Red); the DENV-2 E protein was probed with DENV-2 human antiserum (purified IgG) and anti-human IgG Alexa-633 (Blue). Images were examined using a Zeiss LSM 780 meta confocal 63×objective lens.
    Figure Legend Snippet: AaMCR and AaSR-C function in a pathway that opposes DENV-2 infection. (A) The interaction between 3 AaMCR fragments and AaSR-C in co-IP assays. Three AaMCR gene fragments were cloned into the pMT/BiP/V5-His A vector. The recombinant plasmids were transiently transfected into S2 cells. The cell supernatant was used for investigation of the AaMCR/AaSR-C interaction. The protein complex was pulled down with a rabbit anti-V5 antibody and probed using a mouse anti-HA antibody. The experiment was reproduced 3 times. (B) Expression and purification of AaMCR-a in Drosophila S2 cells. The purified AaMCR-a was separated through SDS-PAGE (Left Panel) and detected with an anti-HA antibody via western blotting (Right Panel). The supernatant from empty vector-transfected S2 cells was used as the mock control. (C) AaSR-C-Ex acted as an adaptor in the interaction between the AaMCR-a and DENV-2 E proteins. The purified AaSR-C-Ex, AaMCR-a and DENV-2 E proteins were mixed and pulled down with a mouse anti-HA antibody (AaMCR-a) and detected using a rabbit anti-V5 antibody (AaSR-C) and anti-FLAG-HRP antibody (DENV-2 E). The experiment was repeated 3 times with similar results. (D) AaSR-C-Ex connected AaMCR-a to DENV-2 virions. Purified AaMCR-a or BSA was pre-coated into the ELISA plate wells. DENV-2 virions either mixed with AaSR-C-Ex or without AaSR-C-Ex were added to the protein-coated wells. The signal was detected using the flavivirus E mAb 4G2. The data are expressed as the mean ± standard error. The experiment was reproduced by 3 times with similar results. (E) Double knockdown of AaMCR and AaSR-C showed similar effects on DENV-2 infection to individual knockdown. Both AaSR-C (i) and AaMCR (ii) were knocked down using a dsRNA mixture in the AaSR-C / AaMCR co-silenced group. DENV-2 replication and the numbers of infectious DENV-2 virions in the mosquitoes were measured via qPCR (iii) and plaque assays (iv). Statistical analysis was performed using the non-parametric Mann-Whitney test. The data on gene silencing (i, ii) and from plaque assays (iv) are expressed as the mean ± standard error. The horizontal line depicts the median (iii). Each dot represents an individual mosquito. The result was representative of 3 independent experiments. (F) Immunostaining of AaMCR, AaSR-C and DENV-2 in A. aegypti hemocytes. The hemocytes were dissected from uninfected mosquitoes, AaMCR and/or AaSR-C silenced infected mosquitoes and GFP dsRNA treated infected mosquitoes at 6 days post-infection. AaSR-C was stained with anti-rabbit IgG Alexa-488 (Green); AaMCR was probed using anti-mouse IgG Alexa-546 (Red); the DENV-2 E protein was probed with DENV-2 human antiserum (purified IgG) and anti-human IgG Alexa-633 (Blue). Images were examined using a Zeiss LSM 780 meta confocal 63×objective lens.

    Techniques Used: Infection, Co-Immunoprecipitation Assay, Clone Assay, Plasmid Preparation, Recombinant, Transfection, Expressing, Purification, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, MANN-WHITNEY, Immunostaining, Staining

    28) Product Images from "A Therapeutic Antiviral Antibody Inhibits the Anterograde Directed Neuron-to-Cell Spread of Herpes Simplex Virus and Protects against Ocular Disease"

    Article Title: A Therapeutic Antiviral Antibody Inhibits the Anterograde Directed Neuron-to-Cell Spread of Herpes Simplex Virus and Protects against Ocular Disease

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2017.02115

    Distribution of mAb hu2c at the initially HSV-1-infected ipsilateral eye. Mice were infected by microinjection of 1 × 10 5 PFU HSV-1 KOS into the anterior chamber of the right eye. MAb hu2c was intravenously applied at 48 h post-infection. Six hours after the antibody injection the ipsilateral eyes were removed. Naïve mice were accordingly treated with mAb hu2c as control. Cross-sections of these eyes were analyzed for HSV infection and the bound humanized antibody mAb hu2c. HSV-infected cells were stained with a mouse-anti-HSV-1/2-gD antibody and an Alexa488-conjugated goat-anti-mouse secondary antibody. MAb hu2c was detected with a Cy3-conjugated goat-anti-human-IgG secondary antibody. Magnification: 25× and 100× as indicated. Arrows indicate the cornea at the front section of the eye.
    Figure Legend Snippet: Distribution of mAb hu2c at the initially HSV-1-infected ipsilateral eye. Mice were infected by microinjection of 1 × 10 5 PFU HSV-1 KOS into the anterior chamber of the right eye. MAb hu2c was intravenously applied at 48 h post-infection. Six hours after the antibody injection the ipsilateral eyes were removed. Naïve mice were accordingly treated with mAb hu2c as control. Cross-sections of these eyes were analyzed for HSV infection and the bound humanized antibody mAb hu2c. HSV-infected cells were stained with a mouse-anti-HSV-1/2-gD antibody and an Alexa488-conjugated goat-anti-mouse secondary antibody. MAb hu2c was detected with a Cy3-conjugated goat-anti-human-IgG secondary antibody. Magnification: 25× and 100× as indicated. Arrows indicate the cornea at the front section of the eye.

    Techniques Used: Infection, Mouse Assay, Injection, Staining

    Distribution of mAb hu2c at the HSV-1-infected contralateral retina. (A) Mice were infected by microinjection of 1 × 10 5 PFU HSV-1 KOS into the anterior chamber of the right eye. The HSV-gB-specific, humanized antiviral antibody mAb hu2c was intravenously applied on days 6, 7, or 8, the time window when the virus is expected to reach the contralateral retina ( Vann and Atherton, 1991 ). Six hours after antibody injection, the eyes were removed and cross-sections of the contralateral retina were analyzed for mAb hu2c. Bound mAb hu2c was stained with a Cy3-conjugated goat-anti-human-IgG secondary antibody. (B) Additionally, cross-sections from eyes removed on day 8 were stained for HSV-1 infection with a mouse-anti-HSV-1/2-gD antibody and an Alexa488-conjugated goat-anti-mouse secondary antibody to verify co-localization of HSV-1 infection and bound mAb hu2c. Scale bars = 50 μM.
    Figure Legend Snippet: Distribution of mAb hu2c at the HSV-1-infected contralateral retina. (A) Mice were infected by microinjection of 1 × 10 5 PFU HSV-1 KOS into the anterior chamber of the right eye. The HSV-gB-specific, humanized antiviral antibody mAb hu2c was intravenously applied on days 6, 7, or 8, the time window when the virus is expected to reach the contralateral retina ( Vann and Atherton, 1991 ). Six hours after antibody injection, the eyes were removed and cross-sections of the contralateral retina were analyzed for mAb hu2c. Bound mAb hu2c was stained with a Cy3-conjugated goat-anti-human-IgG secondary antibody. (B) Additionally, cross-sections from eyes removed on day 8 were stained for HSV-1 infection with a mouse-anti-HSV-1/2-gD antibody and an Alexa488-conjugated goat-anti-mouse secondary antibody to verify co-localization of HSV-1 infection and bound mAb hu2c. Scale bars = 50 μM.

    Techniques Used: Infection, Mouse Assay, Injection, Staining

    29) Product Images from "Complement-Related Proteins Control the Flavivirus Infection of Aedes aegypti by Inducing Antimicrobial Peptides"

    Article Title: Complement-Related Proteins Control the Flavivirus Infection of Aedes aegypti by Inducing Antimicrobial Peptides

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004027

    A Scavenger receptor-C with CCP domains recognizes DENV-2 in vitro and in vivo . (A) Percentage of amino acid identity of insect SR-Cs. (B) Schematic representation of SR-Cs in A. aegypti and D. melanogaster . The functional modules were predicted in SMART ( http://smart.embl-heidelberg.de/smart/set_mode.cgi?GENOMIC=1 ) and Pfam websites ( http://pfam.sanger.ac.uk/ ). (C) Expression and purification of AaSR-C from Drosophila S2 cells. The extracellular region (AaSR-C-Ex) or full length (AaSR-C-Full) AaSR-C was cloned into the pMT/BiP/V5-His-A expression vector. The recombinant plasmids were transfected into Drosophila S2 cells, and their expression was probed using an anti-V5 mAb. The supernatant or lysates from mock-transfected S2 cells was used as the mock control (Left panel). Recombinant AaSR-C-Ex, produced in Drosophila cells, was purified using an Ni-His column (Right panel). (D) AaSR-C-Ex interacted with DENV-2 E proteins in co-immunoprecipitation assay. Purified AaSR-C-Ex (V5) and DENV-2 E (FLAG) were used to investigate the interaction of the proteins. Control rabbit IgG was used as a mock control to exclude non-specific interactions. The protein complex was pulled down with a rabbit anti-FLAG antibody and detected using a mouse anti-V5 antibody. We reproduced the experiment 3 times. (E) AaSR-C-Ex captured DENV-2 virions in an ELISA. Binding was probed using the flavivirus E mAb 4G2. The data are presented as the mean ± standard error. The experiment was reproduced 3 times. (F) AaSR-C bound DENV-2 virions on the cell surface. A Cu 2+ -inducible stable S2 cell line was generated to express the full-length AaSR-C. DENV-2 virions were incubated with AaSR-C expressing cells at 4°C for 1 hr. Non-induced stable cells and empty vector-transfected S2 cells containing virions served as the mock control groups. The interaction between AaSR-C and DENV was measured through flow cytometry. The DENV virions were stained using the flaviviral E mAb 4G2 and anti-mouse IgG Alexa-488; AaSR-C was probed using a Myc mAb and anti-rabbit IgG Phycoerythrin (PE). The data was analyzed using FlowJo software. The presented data was representative of 3 independent experiments with similar results. (G) The in vivo association between AaSR-C and DENV-2 in A.aegypti hemocytes. Hemolymph was collected from uninfected mosquitoes, AaSR-C silenced infected mosquitoes and GFP dsRNA treated infected mosquitoes to undergo immunofluorescence staining. AaSR-C was stained with anti-rabbit IgG Alexa-488 (Green), and the DENV-2 E protein was identified using anti-mouse IgG Alexa-546 (Red). Nuclei were stained blue with To-Pro-3 iodide (Blue). Images were examined using a Zeiss LSM 780 meta confocal 63×objective lens.
    Figure Legend Snippet: A Scavenger receptor-C with CCP domains recognizes DENV-2 in vitro and in vivo . (A) Percentage of amino acid identity of insect SR-Cs. (B) Schematic representation of SR-Cs in A. aegypti and D. melanogaster . The functional modules were predicted in SMART ( http://smart.embl-heidelberg.de/smart/set_mode.cgi?GENOMIC=1 ) and Pfam websites ( http://pfam.sanger.ac.uk/ ). (C) Expression and purification of AaSR-C from Drosophila S2 cells. The extracellular region (AaSR-C-Ex) or full length (AaSR-C-Full) AaSR-C was cloned into the pMT/BiP/V5-His-A expression vector. The recombinant plasmids were transfected into Drosophila S2 cells, and their expression was probed using an anti-V5 mAb. The supernatant or lysates from mock-transfected S2 cells was used as the mock control (Left panel). Recombinant AaSR-C-Ex, produced in Drosophila cells, was purified using an Ni-His column (Right panel). (D) AaSR-C-Ex interacted with DENV-2 E proteins in co-immunoprecipitation assay. Purified AaSR-C-Ex (V5) and DENV-2 E (FLAG) were used to investigate the interaction of the proteins. Control rabbit IgG was used as a mock control to exclude non-specific interactions. The protein complex was pulled down with a rabbit anti-FLAG antibody and detected using a mouse anti-V5 antibody. We reproduced the experiment 3 times. (E) AaSR-C-Ex captured DENV-2 virions in an ELISA. Binding was probed using the flavivirus E mAb 4G2. The data are presented as the mean ± standard error. The experiment was reproduced 3 times. (F) AaSR-C bound DENV-2 virions on the cell surface. A Cu 2+ -inducible stable S2 cell line was generated to express the full-length AaSR-C. DENV-2 virions were incubated with AaSR-C expressing cells at 4°C for 1 hr. Non-induced stable cells and empty vector-transfected S2 cells containing virions served as the mock control groups. The interaction between AaSR-C and DENV was measured through flow cytometry. The DENV virions were stained using the flaviviral E mAb 4G2 and anti-mouse IgG Alexa-488; AaSR-C was probed using a Myc mAb and anti-rabbit IgG Phycoerythrin (PE). The data was analyzed using FlowJo software. The presented data was representative of 3 independent experiments with similar results. (G) The in vivo association between AaSR-C and DENV-2 in A.aegypti hemocytes. Hemolymph was collected from uninfected mosquitoes, AaSR-C silenced infected mosquitoes and GFP dsRNA treated infected mosquitoes to undergo immunofluorescence staining. AaSR-C was stained with anti-rabbit IgG Alexa-488 (Green), and the DENV-2 E protein was identified using anti-mouse IgG Alexa-546 (Red). Nuclei were stained blue with To-Pro-3 iodide (Blue). Images were examined using a Zeiss LSM 780 meta confocal 63×objective lens.

    Techniques Used: In Vitro, In Vivo, Functional Assay, Expressing, Purification, Clone Assay, Plasmid Preparation, Recombinant, Transfection, Produced, Co-Immunoprecipitation Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Generated, Incubation, Flow Cytometry, Cytometry, Staining, Software, Infection, Immunofluorescence

    AaMCR and AaSR-C function in a pathway that opposes DENV-2 infection. (A) The interaction between 3 AaMCR fragments and AaSR-C in co-IP assays. Three AaMCR gene fragments were cloned into the pMT/BiP/V5-His A vector. The recombinant plasmids were transiently transfected into S2 cells. The cell supernatant was used for investigation of the AaMCR/AaSR-C interaction. The protein complex was pulled down with a rabbit anti-V5 antibody and probed using a mouse anti-HA antibody. The experiment was reproduced 3 times. (B) Expression and purification of AaMCR-a in Drosophila S2 cells. The purified AaMCR-a was separated through SDS-PAGE (Left Panel) and detected with an anti-HA antibody via western blotting (Right Panel). The supernatant from empty vector-transfected S2 cells was used as the mock control. (C) AaSR-C-Ex acted as an adaptor in the interaction between the AaMCR-a and DENV-2 E proteins. The purified AaSR-C-Ex, AaMCR-a and DENV-2 E proteins were mixed and pulled down with a mouse anti-HA antibody (AaMCR-a) and detected using a rabbit anti-V5 antibody (AaSR-C) and anti-FLAG-HRP antibody (DENV-2 E). The experiment was repeated 3 times with similar results. (D) AaSR-C-Ex connected AaMCR-a to DENV-2 virions. Purified AaMCR-a or BSA was pre-coated into the ELISA plate wells. DENV-2 virions either mixed with AaSR-C-Ex or without AaSR-C-Ex were added to the protein-coated wells. The signal was detected using the flavivirus E mAb 4G2. The data are expressed as the mean ± standard error. The experiment was reproduced by 3 times with similar results. (E) Double knockdown of AaMCR and AaSR-C showed similar effects on DENV-2 infection to individual knockdown. Both AaSR-C (i) and AaMCR (ii) were knocked down using a dsRNA mixture in the AaSR-C / AaMCR co-silenced group. DENV-2 replication and the numbers of infectious DENV-2 virions in the mosquitoes were measured via qPCR (iii) and plaque assays (iv). Statistical analysis was performed using the non-parametric Mann-Whitney test. The data on gene silencing (i, ii) and from plaque assays (iv) are expressed as the mean ± standard error. The horizontal line depicts the median (iii). Each dot represents an individual mosquito. The result was representative of 3 independent experiments. (F) Immunostaining of AaMCR, AaSR-C and DENV-2 in A. aegypti hemocytes. The hemocytes were dissected from uninfected mosquitoes, AaMCR and/or AaSR-C silenced infected mosquitoes and GFP dsRNA treated infected mosquitoes at 6 days post-infection. AaSR-C was stained with anti-rabbit IgG Alexa-488 (Green); AaMCR was probed using anti-mouse IgG Alexa-546 (Red); the DENV-2 E protein was probed with DENV-2 human antiserum (purified IgG) and anti-human IgG Alexa-633 (Blue). Images were examined using a Zeiss LSM 780 meta confocal 63×objective lens.
    Figure Legend Snippet: AaMCR and AaSR-C function in a pathway that opposes DENV-2 infection. (A) The interaction between 3 AaMCR fragments and AaSR-C in co-IP assays. Three AaMCR gene fragments were cloned into the pMT/BiP/V5-His A vector. The recombinant plasmids were transiently transfected into S2 cells. The cell supernatant was used for investigation of the AaMCR/AaSR-C interaction. The protein complex was pulled down with a rabbit anti-V5 antibody and probed using a mouse anti-HA antibody. The experiment was reproduced 3 times. (B) Expression and purification of AaMCR-a in Drosophila S2 cells. The purified AaMCR-a was separated through SDS-PAGE (Left Panel) and detected with an anti-HA antibody via western blotting (Right Panel). The supernatant from empty vector-transfected S2 cells was used as the mock control. (C) AaSR-C-Ex acted as an adaptor in the interaction between the AaMCR-a and DENV-2 E proteins. The purified AaSR-C-Ex, AaMCR-a and DENV-2 E proteins were mixed and pulled down with a mouse anti-HA antibody (AaMCR-a) and detected using a rabbit anti-V5 antibody (AaSR-C) and anti-FLAG-HRP antibody (DENV-2 E). The experiment was repeated 3 times with similar results. (D) AaSR-C-Ex connected AaMCR-a to DENV-2 virions. Purified AaMCR-a or BSA was pre-coated into the ELISA plate wells. DENV-2 virions either mixed with AaSR-C-Ex or without AaSR-C-Ex were added to the protein-coated wells. The signal was detected using the flavivirus E mAb 4G2. The data are expressed as the mean ± standard error. The experiment was reproduced by 3 times with similar results. (E) Double knockdown of AaMCR and AaSR-C showed similar effects on DENV-2 infection to individual knockdown. Both AaSR-C (i) and AaMCR (ii) were knocked down using a dsRNA mixture in the AaSR-C / AaMCR co-silenced group. DENV-2 replication and the numbers of infectious DENV-2 virions in the mosquitoes were measured via qPCR (iii) and plaque assays (iv). Statistical analysis was performed using the non-parametric Mann-Whitney test. The data on gene silencing (i, ii) and from plaque assays (iv) are expressed as the mean ± standard error. The horizontal line depicts the median (iii). Each dot represents an individual mosquito. The result was representative of 3 independent experiments. (F) Immunostaining of AaMCR, AaSR-C and DENV-2 in A. aegypti hemocytes. The hemocytes were dissected from uninfected mosquitoes, AaMCR and/or AaSR-C silenced infected mosquitoes and GFP dsRNA treated infected mosquitoes at 6 days post-infection. AaSR-C was stained with anti-rabbit IgG Alexa-488 (Green); AaMCR was probed using anti-mouse IgG Alexa-546 (Red); the DENV-2 E protein was probed with DENV-2 human antiserum (purified IgG) and anti-human IgG Alexa-633 (Blue). Images were examined using a Zeiss LSM 780 meta confocal 63×objective lens.

    Techniques Used: Infection, Co-Immunoprecipitation Assay, Clone Assay, Plasmid Preparation, Recombinant, Transfection, Expressing, Purification, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, MANN-WHITNEY, Immunostaining, Staining

    30) Product Images from "Recombinant O-mannosylated protein production (PstS-1) from Mycobacterium tuberculosis in Pichia pastoris (Komagataella phaffii) as a tool to study tuberculosis infection"

    Article Title: Recombinant O-mannosylated protein production (PstS-1) from Mycobacterium tuberculosis in Pichia pastoris (Komagataella phaffii) as a tool to study tuberculosis infection

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-019-1059-3

    Box plot analysis of antibody reactivity to recombinant antigens rPstS-1. Recombinant proteins (from E. coli and P. pastoris ) were tested with PPD-negative (n = 5), PPD-positive sera (n = 5) at 1:100 and active-TB sera (n = 30) at 1:500 dilution. The reactivity is reported as difference of the absorbance units observed for the different group of sera by ELISA using a IgG2 conjugated with AP. Different sera of each group were tested by duplicate. Mean (dashed lane), median, 10th, 25th, 75th and 90th percentiles as vertical boxes with error bars are shown. Dunn´s test ws used for all pairwise comparisons and comparisons against a control group (PBS) following rank-based ANOVA
    Figure Legend Snippet: Box plot analysis of antibody reactivity to recombinant antigens rPstS-1. Recombinant proteins (from E. coli and P. pastoris ) were tested with PPD-negative (n = 5), PPD-positive sera (n = 5) at 1:100 and active-TB sera (n = 30) at 1:500 dilution. The reactivity is reported as difference of the absorbance units observed for the different group of sera by ELISA using a IgG2 conjugated with AP. Different sera of each group were tested by duplicate. Mean (dashed lane), median, 10th, 25th, 75th and 90th percentiles as vertical boxes with error bars are shown. Dunn´s test ws used for all pairwise comparisons and comparisons against a control group (PBS) following rank-based ANOVA

    Techniques Used: Recombinant, Enzyme-linked Immunosorbent Assay

    31) Product Images from "Epstein–Barr Virus Lytic Reactivation Induces IgG4 Production by Host B Lymphocytes in Graves' Disease Patients and Controls: A Subset of Graves' Disease Is an IgG4-Related Disease-Like Condition"

    Article Title: Epstein–Barr Virus Lytic Reactivation Induces IgG4 Production by Host B Lymphocytes in Graves' Disease Patients and Controls: A Subset of Graves' Disease Is an IgG4-Related Disease-Like Condition

    Journal: Viral Immunology

    doi: 10.1089/vim.2018.0042

    EBER1-positive cells and IgG4-positive cells were observed in the same areas. Resected tissues showed the diffuse hyperplasia of thyroid follicular epithelial cells with the focal infiltration of lymphocytes, but not tumefactive lesions, storiform fibrosis, or obliterative phlebitis (A) . EBER1-positive cells and IgG4-positive plasma cells were observed in the same area with lymphoid cell infiltration (B, D) . Six of the seven cases had a large number of IgG4-positive plasma cells (10/HPF
    Figure Legend Snippet: EBER1-positive cells and IgG4-positive cells were observed in the same areas. Resected tissues showed the diffuse hyperplasia of thyroid follicular epithelial cells with the focal infiltration of lymphocytes, but not tumefactive lesions, storiform fibrosis, or obliterative phlebitis (A) . EBER1-positive cells and IgG4-positive plasma cells were observed in the same area with lymphoid cell infiltration (B, D) . Six of the seven cases had a large number of IgG4-positive plasma cells (10/HPF

    Techniques Used:

    Production of IgG and IgG4 during the EBV reactivation period. Culture fluids were sampled on days 0, 5, 10, and 12, and IgG (A) and IgG4 (B) were then measured by ELISA. Time course changes were significant in Friedman's analysis of variance. IgG4 percentages (C) were higher than normal serum levels (approximately 4%). However, no significant difference was observed between patients and controls (D) .
    Figure Legend Snippet: Production of IgG and IgG4 during the EBV reactivation period. Culture fluids were sampled on days 0, 5, 10, and 12, and IgG (A) and IgG4 (B) were then measured by ELISA. Time course changes were significant in Friedman's analysis of variance. IgG4 percentages (C) were higher than normal serum levels (approximately 4%). However, no significant difference was observed between patients and controls (D) .

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Detection of IgG4(+)72A1(+) double-positive cells in culture cells on day 5. We detected IgG4-positive and EBV-reactivated [IgG4(+)72A1(+)] cells in culture cells on day 5 and confirmed sorted cells by confocal laser microscope. Red spots are surface IgG4 and fine green dots ).
    Figure Legend Snippet: Detection of IgG4(+)72A1(+) double-positive cells in culture cells on day 5. We detected IgG4-positive and EBV-reactivated [IgG4(+)72A1(+)] cells in culture cells on day 5 and confirmed sorted cells by confocal laser microscope. Red spots are surface IgG4 and fine green dots ).

    Techniques Used: Microscopy

    32) Product Images from "p53 Represses Class Switch Recombination to IgG2a through its Antioxidant Function"

    Article Title: p53 Represses Class Switch Recombination to IgG2a through its Antioxidant Function

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.0904085

    Increased ROS in switching p53−/− splenic B cells; NAC inhibits CSR and abolishes increased IgG2a switching in p53−/− splenic B cells (A) Shown are overlay histograms of wild-type (red profile) and p53−/− (blue profile) splenic B cells loaded with the CM-H 2 -DCFDA fluorescent ROS probe after activation with LPS + anti-δ-dextran + IFNγ for 48 h. Treatment with 10 mM (blue profile) and 20 mM NAC (green profile) decreases intracellular ROS in activated wild-type and p53−/− splenic B cells. (B) CFSE-loaded cells were activated for IgG2a switching and treated with 10 mM NAC. IgG2a switching was assessed by flow cytometry for surface Ig staining after 2.5 days. Representative FACS plots of 3 independent experiments are shown. Percentages of IgG2a switched cells are given within the gates. (C) CFSE-loaded wild-type splenic B cells were activated with LPS + cytokines and/or anti-δ-dextran to induce CSR to the indicated Ig isotypes. CSR was assessed by flow cytometry for surface Ig staining. Percentages of switched cells are indicated within the gates. Top row shows untreated control cultures, middle row shows cultures treated with 10 mM NAC. Bottom row shows overlay histograms of CSFE fluorescence in control cultures (red profiles) and 10 mM NAC treated cultures (blue profiles). Representative FACS plots of three experiments are shown. (D) Bar graph shows data from the 3 experiments normalized to untreated cells, shown as 100%. Means (+SEM) of percentages of switched cells in NAC-treated cutures relative to untreated cultures are shown. Significance was determined by the one-sample t test. (E) Sμ LM-PCR was performed on threefold dilutions of Gapdh-normalized DNA from wild-type and aid−/− splenic B cells activated with LPS + anti-δ-dextran with or without 10 mM NAC for 2 days. LM-PCR products were blotted and hybridized with an internal Sμ probe. Densitometry scanning was performed on all three lanes representing the threefold dilutions. Untreated sample was set at 1.0 to determine relative density. Representative result of 2 experiments is shown.
    Figure Legend Snippet: Increased ROS in switching p53−/− splenic B cells; NAC inhibits CSR and abolishes increased IgG2a switching in p53−/− splenic B cells (A) Shown are overlay histograms of wild-type (red profile) and p53−/− (blue profile) splenic B cells loaded with the CM-H 2 -DCFDA fluorescent ROS probe after activation with LPS + anti-δ-dextran + IFNγ for 48 h. Treatment with 10 mM (blue profile) and 20 mM NAC (green profile) decreases intracellular ROS in activated wild-type and p53−/− splenic B cells. (B) CFSE-loaded cells were activated for IgG2a switching and treated with 10 mM NAC. IgG2a switching was assessed by flow cytometry for surface Ig staining after 2.5 days. Representative FACS plots of 3 independent experiments are shown. Percentages of IgG2a switched cells are given within the gates. (C) CFSE-loaded wild-type splenic B cells were activated with LPS + cytokines and/or anti-δ-dextran to induce CSR to the indicated Ig isotypes. CSR was assessed by flow cytometry for surface Ig staining. Percentages of switched cells are indicated within the gates. Top row shows untreated control cultures, middle row shows cultures treated with 10 mM NAC. Bottom row shows overlay histograms of CSFE fluorescence in control cultures (red profiles) and 10 mM NAC treated cultures (blue profiles). Representative FACS plots of three experiments are shown. (D) Bar graph shows data from the 3 experiments normalized to untreated cells, shown as 100%. Means (+SEM) of percentages of switched cells in NAC-treated cutures relative to untreated cultures are shown. Significance was determined by the one-sample t test. (E) Sμ LM-PCR was performed on threefold dilutions of Gapdh-normalized DNA from wild-type and aid−/− splenic B cells activated with LPS + anti-δ-dextran with or without 10 mM NAC for 2 days. LM-PCR products were blotted and hybridized with an internal Sμ probe. Densitometry scanning was performed on all three lanes representing the threefold dilutions. Untreated sample was set at 1.0 to determine relative density. Representative result of 2 experiments is shown.

    Techniques Used: Activation Assay, Flow Cytometry, Cytometry, Staining, FACS, Fluorescence, Polymerase Chain Reaction

    Polyoma virus infected p53−/− mice show increased in vivo IgG2a switching p53−/− , aid−/− , and wild-type littermate mice were inoculated i.p. with 2×10 6 pfu of polyoma virus strain A2. Splenic germinal center B cells were analyzed for surface isotype expression by flow cytometry 12–20 days after inoculation. (A) FACS plots show surface Ig expression on B220+ GL7+ splenic B cells from an experiment in which cells were harvested 14 days after infection. Percentages of switched cells are depicted next to the gates. (B) Data points and bars represent individual mice and means (n=7 for wild-type, n=5 for p53−/− ) of the percentages of isotype switched cells within the B220+ GL7+ splenic germinal center B-cell subset. Three independent experiments were performed using: 3, 2, and 2 wild-type mice, 1, 2, and 2 p53−/− mice, and in the second experiment (shown) 2 aid−/− mice were included. Significance was determined by the Mann-Whitney U test.
    Figure Legend Snippet: Polyoma virus infected p53−/− mice show increased in vivo IgG2a switching p53−/− , aid−/− , and wild-type littermate mice were inoculated i.p. with 2×10 6 pfu of polyoma virus strain A2. Splenic germinal center B cells were analyzed for surface isotype expression by flow cytometry 12–20 days after inoculation. (A) FACS plots show surface Ig expression on B220+ GL7+ splenic B cells from an experiment in which cells were harvested 14 days after infection. Percentages of switched cells are depicted next to the gates. (B) Data points and bars represent individual mice and means (n=7 for wild-type, n=5 for p53−/− ) of the percentages of isotype switched cells within the B220+ GL7+ splenic germinal center B-cell subset. Three independent experiments were performed using: 3, 2, and 2 wild-type mice, 1, 2, and 2 p53−/− mice, and in the second experiment (shown) 2 aid−/− mice were included. Significance was determined by the Mann-Whitney U test.

    Techniques Used: Infection, Mouse Assay, In Vivo, Expressing, Flow Cytometry, Cytometry, FACS, MANN-WHITNEY

    S region DSBs are increased p53−/− splenic B cells induced for IgG2a switching Sμ and Sγ2a LM-PCR were performed on threefold dilutions of GAPDH-normalized DNA isolated from wild-type (WT), p53−/− , and aid−/− splenic B cells that had been stimulated for 2 days with LPS+IL4 or LPS+IFNγ. Blunt DSBs in Sμ and Sγ2a were assessed by use of 5’ Sμ or 5’ Sγ2a primer, respectively, in combination with a linker specific primer. PCR products were blotted and hybridized with an internal Sμ or Sγ2a probe, respectively. (A) Sμ DSBs in cells activated for IgG1 switching. (B) Sμ DSBs in cells activated for IgG2a switching. (C) Sγ2a DSBs in cells activated for IgG2a switching. (D) Bar graph depicts means (+SEM) of densitometry measurements of autoradiographic films, normalized to WT, which was set at 1.0 (n=4 for all experiments, except for Sγ2a DSBs under IgG2a conditions, n=3.) All 3 titration lanes were scanned together. Each replicate experiment was performed on material from a separate set of mice. Two independent experiments using two sets of mice were performed. (E) Mutations in Sμ-Sγ3 junctions from WT and p53−/− splenic B cells activated to undergo IgG3 switching. Mutation frequency per 50-nucleotide segment is plotted for WT (black bars) and p53−/− (white bars). Overall mutation frequency is 3.1-fold increased in p53−/− versus WT (p = 0.00001). Total mutations/nucleotides analyzed for WT: 31/10,231; for p53: 30/3,182.
    Figure Legend Snippet: S region DSBs are increased p53−/− splenic B cells induced for IgG2a switching Sμ and Sγ2a LM-PCR were performed on threefold dilutions of GAPDH-normalized DNA isolated from wild-type (WT), p53−/− , and aid−/− splenic B cells that had been stimulated for 2 days with LPS+IL4 or LPS+IFNγ. Blunt DSBs in Sμ and Sγ2a were assessed by use of 5’ Sμ or 5’ Sγ2a primer, respectively, in combination with a linker specific primer. PCR products were blotted and hybridized with an internal Sμ or Sγ2a probe, respectively. (A) Sμ DSBs in cells activated for IgG1 switching. (B) Sμ DSBs in cells activated for IgG2a switching. (C) Sγ2a DSBs in cells activated for IgG2a switching. (D) Bar graph depicts means (+SEM) of densitometry measurements of autoradiographic films, normalized to WT, which was set at 1.0 (n=4 for all experiments, except for Sγ2a DSBs under IgG2a conditions, n=3.) All 3 titration lanes were scanned together. Each replicate experiment was performed on material from a separate set of mice. Two independent experiments using two sets of mice were performed. (E) Mutations in Sμ-Sγ3 junctions from WT and p53−/− splenic B cells activated to undergo IgG3 switching. Mutation frequency per 50-nucleotide segment is plotted for WT (black bars) and p53−/− (white bars). Overall mutation frequency is 3.1-fold increased in p53−/− versus WT (p = 0.00001). Total mutations/nucleotides analyzed for WT: 31/10,231; for p53: 30/3,182.

    Techniques Used: Polymerase Chain Reaction, Isolation, Titration, Mouse Assay, Mutagenesis

    p53 Ser18 phosphorylation regulates IgG2a switching CFSE-loaded splenic B cells from p53S18A and wild-type control mice were cultured for 2.5 days with LPS, cytokines and/or anti-δ-dextran to induce Ig class switching to the indicated isotypes. Class switching was determined by flow cytometry for surface Ig staining. (A) Representative FACS plots are shown. Upper panel shows equal CFSE loading in wild-type mice from the same colony and p53S18A splenic B cells. Percentages of switched cells are indicated within the gates. (B) Bar graph depicts data from 4 sets of mice, which were all analyzed in one experiment. Percent CSR in p53S18A relative to WT (+SEM) for the different isotypes are shown. Significance was determined by the one-sample t test.
    Figure Legend Snippet: p53 Ser18 phosphorylation regulates IgG2a switching CFSE-loaded splenic B cells from p53S18A and wild-type control mice were cultured for 2.5 days with LPS, cytokines and/or anti-δ-dextran to induce Ig class switching to the indicated isotypes. Class switching was determined by flow cytometry for surface Ig staining. (A) Representative FACS plots are shown. Upper panel shows equal CFSE loading in wild-type mice from the same colony and p53S18A splenic B cells. Percentages of switched cells are indicated within the gates. (B) Bar graph depicts data from 4 sets of mice, which were all analyzed in one experiment. Percent CSR in p53S18A relative to WT (+SEM) for the different isotypes are shown. Significance was determined by the one-sample t test.

    Techniques Used: Mouse Assay, Cell Culture, Flow Cytometry, Cytometry, Staining, FACS

    p53-deficient splenic B cells show increased IgG2a switching in culture CFSE-loaded splenic B cells from p53−/− and wild-type littermate mice were cultured for 2.5 days with LPS, cytokines and/or anti-δ-dextran to induce Ig class switching to the indicated isotypes. (A) Cells were analyzed for CSFE-dilution and surface Ig by flow cytometry; representative FACS plots are shown; percentages of switched cells are indicated within the gates. (B) Data from 8 experiments (8 sets of mice) were normalized to the percent of wild-type littermate (WT) switching, indicated as 100%. Mean percentages of switching in p53−/− relative to WT (+SEM) for the different isotypes are shown. Significance was determined by the one-sample t test.
    Figure Legend Snippet: p53-deficient splenic B cells show increased IgG2a switching in culture CFSE-loaded splenic B cells from p53−/− and wild-type littermate mice were cultured for 2.5 days with LPS, cytokines and/or anti-δ-dextran to induce Ig class switching to the indicated isotypes. (A) Cells were analyzed for CSFE-dilution and surface Ig by flow cytometry; representative FACS plots are shown; percentages of switched cells are indicated within the gates. (B) Data from 8 experiments (8 sets of mice) were normalized to the percent of wild-type littermate (WT) switching, indicated as 100%. Mean percentages of switching in p53−/− relative to WT (+SEM) for the different isotypes are shown. Significance was determined by the one-sample t test.

    Techniques Used: Mouse Assay, Cell Culture, Flow Cytometry, Cytometry, FACS

    33) Product Images from "The chimeric antibody chLpMab-7 targeting human podoplanin suppresses pulmonary metastasis via ADCC and CDC rather than via its neutralizing activity"

    Article Title: The chimeric antibody chLpMab-7 targeting human podoplanin suppresses pulmonary metastasis via ADCC and CDC rather than via its neutralizing activity

    Journal: Oncotarget

    doi:

    ADCC and CDC activities of chLpMab-7 A. ADCC activities induced by human MNC against hPDPN-expressing cell lines were determined using a 6 h 51 Cr release assay at the E/T ratio of 100 in the presence of 1 μg/ml chLpMab-7 and human IgG. B. CDC activities against hPDPN-expressing cell lines were demonstrated by 51 Cr release assay. * P
    Figure Legend Snippet: ADCC and CDC activities of chLpMab-7 A. ADCC activities induced by human MNC against hPDPN-expressing cell lines were determined using a 6 h 51 Cr release assay at the E/T ratio of 100 in the presence of 1 μg/ml chLpMab-7 and human IgG. B. CDC activities against hPDPN-expressing cell lines were demonstrated by 51 Cr release assay. * P

    Techniques Used: Expressing, Release Assay

    Suppression of experimental lung metastasis by chLpMab-7 CHO/hPDPN cells (5 × 10 5 cells/100 μl) were injected intravenously into nude mice. Animals were assigned to 4 groups, which received chLpMab-7 or human IgG concomitantly with the cell injection (Day 0), chLpMab-7 treatment 1 day after tumor cell injection (Day 1), or chLpMab-7 treatment 5 days after cell injection (Day 5). A. Visible lung metastasis. B. Incidence and number of visible metastases per lung. C. Lung weight. D. Body weight. *** p
    Figure Legend Snippet: Suppression of experimental lung metastasis by chLpMab-7 CHO/hPDPN cells (5 × 10 5 cells/100 μl) were injected intravenously into nude mice. Animals were assigned to 4 groups, which received chLpMab-7 or human IgG concomitantly with the cell injection (Day 0), chLpMab-7 treatment 1 day after tumor cell injection (Day 1), or chLpMab-7 treatment 5 days after cell injection (Day 5). A. Visible lung metastasis. B. Incidence and number of visible metastases per lung. C. Lung weight. D. Body weight. *** p

    Techniques Used: Injection, Mouse Assay

    Anti-tumor effect of chLpMab-7 against PC-10 xenografts PC-10 cells (5 × 10 6 cells/100 μl) were inoculated subcutaneously into BALB/c nude mice. After one day, 100 μg of chLpMab-7 or human IgG antibodies was injected into the peritoneal cavity of mice. The antibodies were injected once per week (human IgG group: n = 6; chLpMab-7 group: n = 6). Human NK cells were injected around the tumors at Day 29 and Day 36. A. Primary tumor growth in human IgG- or chLpMab-7-treated mice. * p
    Figure Legend Snippet: Anti-tumor effect of chLpMab-7 against PC-10 xenografts PC-10 cells (5 × 10 6 cells/100 μl) were inoculated subcutaneously into BALB/c nude mice. After one day, 100 μg of chLpMab-7 or human IgG antibodies was injected into the peritoneal cavity of mice. The antibodies were injected once per week (human IgG group: n = 6; chLpMab-7 group: n = 6). Human NK cells were injected around the tumors at Day 29 and Day 36. A. Primary tumor growth in human IgG- or chLpMab-7-treated mice. * p

    Techniques Used: Mouse Assay, Injection

    Anti-tumor effect of chLpMab-7 on primary tumor development and spontaneous lung metastasis in nude mice inoculated with hPDPN-expressing cells CHO/hPDPN cells (3 × 10 6 cells/100 μl) were inoculated subcutaneously into BALB/c nude mice. After one day, 100 μg of chLpMab-7 or human IgG antibodies was injected into the peritoneal cavity of mice. The antibodies were injected once per week for three weeks (human IgG group: n = 10; chLpMab-7 group: n = 10). A. Comparison of the tumor size and tumor incidence in nude mice (day 30). B. Primary tumor growth in human IgG- or chLpMab-7-treated mice. *** p
    Figure Legend Snippet: Anti-tumor effect of chLpMab-7 on primary tumor development and spontaneous lung metastasis in nude mice inoculated with hPDPN-expressing cells CHO/hPDPN cells (3 × 10 6 cells/100 μl) were inoculated subcutaneously into BALB/c nude mice. After one day, 100 μg of chLpMab-7 or human IgG antibodies was injected into the peritoneal cavity of mice. The antibodies were injected once per week for three weeks (human IgG group: n = 10; chLpMab-7 group: n = 10). A. Comparison of the tumor size and tumor incidence in nude mice (day 30). B. Primary tumor growth in human IgG- or chLpMab-7-treated mice. *** p

    Techniques Used: Mouse Assay, Expressing, Injection

    34) Product Images from "Viral Semaphorin Inhibits Dendritic Cell Phagocytosis and Migration but Is Not Essential for Gammaherpesvirus-Induced Lymphoproliferation in Malignant Catarrhal Fever"

    Article Title: Viral Semaphorin Inhibits Dendritic Cell Phagocytosis and Migration but Is Not Essential for Gammaherpesvirus-Induced Lymphoproliferation in Malignant Catarrhal Fever

    Journal: Journal of Virology

    doi: 10.1128/JVI.03634-14

    A3 is an early gene encoding a 93-kDa glycoprotein secreted during virus infection. (A) Determination of the A3 kinetic class of transcription. BT cells were infected with AlHV-1 and treated with CHX or PAA or left untreated (−). At 24 h p.i., the expression of ORF73 (IE gene), ORF09 (DPOL, E gene), ORF22 (gH, L gene), or A3 was determined by using a reverse transcription-PCR approach, as described in Materials and Methods. (B) Recombineering methodology used to insert a carboxy-terminal hIgG1 Fc fragment tag to the A3 gene and produce the BAC A3Fc strain. (C) The BAC constructs were analyzed by Southern blotting after EcoRI restriction. Probes are indicated: galK, entire galK coding sequence; A3, entire A3 coding sequence. (D) AlHV-sema kinetics of expression after infection with the BAC A3Fc virus (MOI = 0.01). Infected BT cells were fixed and permeabilized at the given time points postinfection before staining with Alexa Fluor 488-nm anti-human IgG1 goat polyserum for AlHV-sema-Fc detection or MAb 15-A primary antibody (specific to gp115 viral complex), followed by Alexa Fluor 488-nm goat anti-mouse IgG secondary antibody. (E) Supernatants were collected and analyzed by immunoblotting with anti-human IgG1 polyserum.
    Figure Legend Snippet: A3 is an early gene encoding a 93-kDa glycoprotein secreted during virus infection. (A) Determination of the A3 kinetic class of transcription. BT cells were infected with AlHV-1 and treated with CHX or PAA or left untreated (−). At 24 h p.i., the expression of ORF73 (IE gene), ORF09 (DPOL, E gene), ORF22 (gH, L gene), or A3 was determined by using a reverse transcription-PCR approach, as described in Materials and Methods. (B) Recombineering methodology used to insert a carboxy-terminal hIgG1 Fc fragment tag to the A3 gene and produce the BAC A3Fc strain. (C) The BAC constructs were analyzed by Southern blotting after EcoRI restriction. Probes are indicated: galK, entire galK coding sequence; A3, entire A3 coding sequence. (D) AlHV-sema kinetics of expression after infection with the BAC A3Fc virus (MOI = 0.01). Infected BT cells were fixed and permeabilized at the given time points postinfection before staining with Alexa Fluor 488-nm anti-human IgG1 goat polyserum for AlHV-sema-Fc detection or MAb 15-A primary antibody (specific to gp115 viral complex), followed by Alexa Fluor 488-nm goat anti-mouse IgG secondary antibody. (E) Supernatants were collected and analyzed by immunoblotting with anti-human IgG1 polyserum.

    Techniques Used: Infection, Expressing, Polymerase Chain Reaction, BAC Assay, Construct, Southern Blot, Sequencing, Staining

    AlHV-sema cell surface binding. (A) Flow cytometry analysis of AlHV-sema-Fc (500 nM) binding to the cell surface of mouse bone marrow-derived macrophages (BMDM), bovine macrophage (BoMac) and bovine turbinate (BT) cell lines, rabbit kidney cells (RK13), wildebeest, bovine, and rabbit CD14 + blood monocytes, bovine monocyte-derived DCs (moDCs), and sheep CD1b + lymph DCs. Purified human IgG1 (500 nM; Calbiochem) and PBS only were used as negative controls. Dendritic and macrophage cell lines were blocked with purified goat IgG (0.5 μg/ml). Staining was revealed using Alexa Fluor 488-nm goat anti-human IgG polyserum. The median fluorescence intensities are indicated for each condition. (B) Confocal analysis of AlHV-sema-Fc and -V5 staining (50 nM) at the surfaces of BT cells. PKH26 counterstaining is shown.
    Figure Legend Snippet: AlHV-sema cell surface binding. (A) Flow cytometry analysis of AlHV-sema-Fc (500 nM) binding to the cell surface of mouse bone marrow-derived macrophages (BMDM), bovine macrophage (BoMac) and bovine turbinate (BT) cell lines, rabbit kidney cells (RK13), wildebeest, bovine, and rabbit CD14 + blood monocytes, bovine monocyte-derived DCs (moDCs), and sheep CD1b + lymph DCs. Purified human IgG1 (500 nM; Calbiochem) and PBS only were used as negative controls. Dendritic and macrophage cell lines were blocked with purified goat IgG (0.5 μg/ml). Staining was revealed using Alexa Fluor 488-nm goat anti-human IgG polyserum. The median fluorescence intensities are indicated for each condition. (B) Confocal analysis of AlHV-sema-Fc and -V5 staining (50 nM) at the surfaces of BT cells. PKH26 counterstaining is shown.

    Techniques Used: Binding Assay, Flow Cytometry, Cytometry, Derivative Assay, Purification, Staining, Fluorescence

    35) Product Images from "Evidence for Fungal Infection in Cerebrospinal Fluid and Brain Tissue from Patients with Amyotrophic Lateral Sclerosis"

    Article Title: Evidence for Fungal Infection in Cerebrospinal Fluid and Brain Tissue from Patients with Amyotrophic Lateral Sclerosis

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.11084

    Immunohistochemistry analysis of brain sections from the frontal cortex of ALS patients. Brain sections (frontal cortex) from ALS patients 9, 10 and 11 were observed with a confocal laser scanning microscope. Sections were obtained from fixed tissue and immunohistochemistry analyses were carried out. Sections were incubated with anti- C. glabrata antibodies (1:500 dilution), followed by incubation with secondary antibody donkey anti-rabbit IgG conjugated to Alexa 555 (1:500). Subsequently, sections were incubated with DAPI (1 μg/ml). The different panels in the figure are indicated. Scale bar: 10 μm.
    Figure Legend Snippet: Immunohistochemistry analysis of brain sections from the frontal cortex of ALS patients. Brain sections (frontal cortex) from ALS patients 9, 10 and 11 were observed with a confocal laser scanning microscope. Sections were obtained from fixed tissue and immunohistochemistry analyses were carried out. Sections were incubated with anti- C. glabrata antibodies (1:500 dilution), followed by incubation with secondary antibody donkey anti-rabbit IgG conjugated to Alexa 555 (1:500). Subsequently, sections were incubated with DAPI (1 μg/ml). The different panels in the figure are indicated. Scale bar: 10 μm.

    Techniques Used: Immunohistochemistry, Laser-Scanning Microscopy, Incubation

    36) Product Images from "Detection of Mycoplasma genitalium-Reactive Cervicovaginal Antibodies among Infected Women ▿"

    Article Title: Detection of Mycoplasma genitalium-Reactive Cervicovaginal Antibodies among Infected Women ▿

    Journal: Clinical and Vaccine Immunology : CVI

    doi: 10.1128/CVI.05174-11

    M. genitalium infection elicits IgG1 and IgG3 subtypes. A subset of strongly IgG-positive samples was characterized further using subtype-specific secondary antibodies. The results from two IgG-positive cases are shown. Cervical and vaginal samples for
    Figure Legend Snippet: M. genitalium infection elicits IgG1 and IgG3 subtypes. A subset of strongly IgG-positive samples was characterized further using subtype-specific secondary antibodies. The results from two IgG-positive cases are shown. Cervical and vaginal samples for

    Techniques Used: Infection

    37) Product Images from "Autoimmunity in Membranous Nephropathy Targets Aldose Reductase and SOD2"

    Article Title: Autoimmunity in Membranous Nephropathy Targets Aldose Reductase and SOD2

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2008121259

    Glomerular eluates from MN patients contain antibodies against AR and SOD2. (A) Glomeruli showed presence of specific IgG 4 antibodies against AR and SOD2. IgGs were also present at a lower titer. Controls were incubated with human serum albumin (HSA)
    Figure Legend Snippet: Glomerular eluates from MN patients contain antibodies against AR and SOD2. (A) Glomeruli showed presence of specific IgG 4 antibodies against AR and SOD2. IgGs were also present at a lower titer. Controls were incubated with human serum albumin (HSA)

    Techniques Used: Incubation

    AR and SOD2 co-localize with IgG4 in glomeruli of MN patient biopsies. (A, B, D, and E) Confocal images of a renal biopsy specimen from a patient with MN. Double IF staining was evaluated for AR (A), SOD2 (D) and IgG 4 (B and E). Merged images are reported
    Figure Legend Snippet: AR and SOD2 co-localize with IgG4 in glomeruli of MN patient biopsies. (A, B, D, and E) Confocal images of a renal biopsy specimen from a patient with MN. Double IF staining was evaluated for AR (A), SOD2 (D) and IgG 4 (B and E). Merged images are reported

    Techniques Used: Staining

    Glomerular eluates from other pathologies do not contain antibodies against AR or SOD2. Dot blot of eluates from microdissected glomeruli of a normal kidney (N) and patients ( n = 7) with other glomerulonephritis implying IgG deposition in glomeruli: Systemic
    Figure Legend Snippet: Glomerular eluates from other pathologies do not contain antibodies against AR or SOD2. Dot blot of eluates from microdissected glomeruli of a normal kidney (N) and patients ( n = 7) with other glomerulonephritis implying IgG deposition in glomeruli: Systemic

    Techniques Used: Dot Blot

    38) Product Images from "A New and Robust Method of Tethering IgG Surrogate Antigens on Lipid Bilayer Membranes to Facilitate the TIRFM Based Live Cell and Single Molecule Imaging Experiments"

    Article Title: A New and Robust Method of Tethering IgG Surrogate Antigens on Lipid Bilayer Membranes to Facilitate the TIRFM Based Live Cell and Single Molecule Imaging Experiments

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0063735

    H12-D-domain mediated PLB membranes only tether whole IgG, but not Fab, F(ab’) 2 or IgM molecules. (A) Shown are representative TIRFM images of Alexa 568-conjugated goat whole IgG, F(ab’) 2 or Fab fragments of goat IgG molecules, or the biotin-conjugated mouse IgM molecules tethered on the surface of PLB membranes with pre-attached H12-D-domain. The PLB membranes tethering the biotin-conjugated mouse IgM molecules were further incubated with the Alexa 568-conjugated streptavidin for TIRFM imaging. Bar is 1.5 µm. (B) Statistical quantification for the mFI of Alexa 568-conjugated goat whole IgG, F(ab’) 2 or Fab fragments of goat IgG molecules tethered on the surface of PLB membranes with pre-attached H12-D-domain. (C) Statistical quantification for the mFI of biotin-conjugated mouse IgM molecules tethered on the surface of PLB membranes with or without pre-attached H12-D-domain. The PLB membranes tethering the biotin-conjugated mouse IgM molecules were further incubated with the Alexa 568-conjugated streptavidin for TIRFM imaging. In both B and C, each dot represents a single measurement for the mFI of the tethered IgG surrogate antigens by Image J software. Bars represent means±SD. Two-tailed t tests were performed for statistical comparisons.
    Figure Legend Snippet: H12-D-domain mediated PLB membranes only tether whole IgG, but not Fab, F(ab’) 2 or IgM molecules. (A) Shown are representative TIRFM images of Alexa 568-conjugated goat whole IgG, F(ab’) 2 or Fab fragments of goat IgG molecules, or the biotin-conjugated mouse IgM molecules tethered on the surface of PLB membranes with pre-attached H12-D-domain. The PLB membranes tethering the biotin-conjugated mouse IgM molecules were further incubated with the Alexa 568-conjugated streptavidin for TIRFM imaging. Bar is 1.5 µm. (B) Statistical quantification for the mFI of Alexa 568-conjugated goat whole IgG, F(ab’) 2 or Fab fragments of goat IgG molecules tethered on the surface of PLB membranes with pre-attached H12-D-domain. (C) Statistical quantification for the mFI of biotin-conjugated mouse IgM molecules tethered on the surface of PLB membranes with or without pre-attached H12-D-domain. The PLB membranes tethering the biotin-conjugated mouse IgM molecules were further incubated with the Alexa 568-conjugated streptavidin for TIRFM imaging. In both B and C, each dot represents a single measurement for the mFI of the tethered IgG surrogate antigens by Image J software. Bars represent means±SD. Two-tailed t tests were performed for statistical comparisons.

    Techniques Used: Incubation, Imaging, Software, Two Tailed Test

    H12-D-domain construct efficiently tethers the IgG surrogate antigens on PLB membranes and these tethered IgG surrogate antigens induce the formation of BCR and surrogate antigen microclusters. (A) Shown are representative TIRFM images of Alexa 647-conjugated goat IgG anti human IgM surrogate antigens tethered on the surface of PLB membranes through H12-D-domain. The Alexa 647-conjugated goat IgG anti human IgM surrogate molecules were pre-incubated (100 or 0 nM) with the PLB membranes containing H12-D-domain. Bar is 1.5 µm. (B) Statistical quantification for the mean fluorescence intensity (mFI) of Alexa 647-conjugated goat IgG anti human IgM surrogate antigens tethered on the surface of PLB membranes. Each dot represents a single measurement for the mFI of the tethered IgG surrogate antigens by Image J software. Bars represent means ± SD. Two-tailed t tests were performed for statistical comparisons. (C) Shown are representative two-color TIRFM images of BCR (green) or the surrogate antigen (red) microclusters within the contact interface of human Ramos B cell with the PLB membranes tethering the Alexa 647-conjugated goat IgG anti human IgM surrogate antigens. Also shown are the merged images. Bar is 1.5 µm. (D) Statistical quantification for the mFI of BCR microclusters (top panel) or surrogate antigen microclusters (lower panel) within the B cell immunological synapse. Each dot shows one measurement from a single cell. Bars represent means ± SD. Two-tailed t tests were performed for statistical comparisons.
    Figure Legend Snippet: H12-D-domain construct efficiently tethers the IgG surrogate antigens on PLB membranes and these tethered IgG surrogate antigens induce the formation of BCR and surrogate antigen microclusters. (A) Shown are representative TIRFM images of Alexa 647-conjugated goat IgG anti human IgM surrogate antigens tethered on the surface of PLB membranes through H12-D-domain. The Alexa 647-conjugated goat IgG anti human IgM surrogate molecules were pre-incubated (100 or 0 nM) with the PLB membranes containing H12-D-domain. Bar is 1.5 µm. (B) Statistical quantification for the mean fluorescence intensity (mFI) of Alexa 647-conjugated goat IgG anti human IgM surrogate antigens tethered on the surface of PLB membranes. Each dot represents a single measurement for the mFI of the tethered IgG surrogate antigens by Image J software. Bars represent means ± SD. Two-tailed t tests were performed for statistical comparisons. (C) Shown are representative two-color TIRFM images of BCR (green) or the surrogate antigen (red) microclusters within the contact interface of human Ramos B cell with the PLB membranes tethering the Alexa 647-conjugated goat IgG anti human IgM surrogate antigens. Also shown are the merged images. Bar is 1.5 µm. (D) Statistical quantification for the mFI of BCR microclusters (top panel) or surrogate antigen microclusters (lower panel) within the B cell immunological synapse. Each dot shows one measurement from a single cell. Bars represent means ± SD. Two-tailed t tests were performed for statistical comparisons.

    Techniques Used: Construct, Incubation, Fluorescence, Software, Two Tailed Test

    The IgG surrogate antigens tethered by H12-D-domain show more uniform distribution with better lateral mobility than the ones tethered by streptavidin. (A) Shown is the distribution of fluorescence intensities (FI) of 10905 IgG surrogate antigen molecules tethered by H12-D-domain (red color) versus 13323 IgG surrogate antigen molecule tethered by streptavidin (blue color) on PLB membranes with a Gaussian fit (red or blue colored curve respectively) to the histogram plot. (B) Cumulative diffusion probability plot of all the calculated instant diffusion coefficients of 10905 IgG surrogate antigen molecules tethered by H12-D-domain (red color) versus 13323 IgG surrogate antigen molecule tethered by streptavidin (blue color) on PLB membranes.
    Figure Legend Snippet: The IgG surrogate antigens tethered by H12-D-domain show more uniform distribution with better lateral mobility than the ones tethered by streptavidin. (A) Shown is the distribution of fluorescence intensities (FI) of 10905 IgG surrogate antigen molecules tethered by H12-D-domain (red color) versus 13323 IgG surrogate antigen molecule tethered by streptavidin (blue color) on PLB membranes with a Gaussian fit (red or blue colored curve respectively) to the histogram plot. (B) Cumulative diffusion probability plot of all the calculated instant diffusion coefficients of 10905 IgG surrogate antigen molecules tethered by H12-D-domain (red color) versus 13323 IgG surrogate antigen molecule tethered by streptavidin (blue color) on PLB membranes.

    Techniques Used: Diffusion-based Assay

    IgG surrogate antigens tethered by H12-D-domain enhance the accumulation of BCR and pSyk into B cell immunological synapse than the ones tethered by streptavidin. (A) Statistical quantification for the mean fluorescence intensity (mFI) of biotin and Alexa 568-conjugated goat IgG anti human IgM surrogate antigens tethered on the surface of PLB membranes by either H12-D-domain (red color) or streptavidin (blue color). Each dot represents a single measurement for the mFI of the tethered IgG surrogate antigens by Image J software. Bars represent means ± SD. Two-tailed t tests were performed for statistical comparisons. (B) Statistical quantification for the accumulation of human IgM-BCRs into the immunological synapse as measured by the mFI of BCR within the immunological synapse from Ramos human B cells that were placed on PLB membranes presenting the same amount of IgG surrogate antigen as shown in A that were tethered by either H12-D-domain or streptavidin. Each dot represents a single measurement for the mFI of IgM-BCRs by Image J software. Bars represent means ± SD. Two-tailed t tests were performed for statistical comparisons. (C) Statistical quantification for the accumulation of pSyk signaling molecules into the immunological synapse as measured by the mFI of BCR within the immunological synapse from Ramos human B cells that were placed on PLB membranes presenting the same amount of IgG surrogate antigen as shown in A that were tethered by either H12-D-domain or streptavidin. Each dot represents a single measurement for the mFI of pSyk by Image J software. Bars represent means ± SD. Two-tailed t tests were performed for statistical comparisons.
    Figure Legend Snippet: IgG surrogate antigens tethered by H12-D-domain enhance the accumulation of BCR and pSyk into B cell immunological synapse than the ones tethered by streptavidin. (A) Statistical quantification for the mean fluorescence intensity (mFI) of biotin and Alexa 568-conjugated goat IgG anti human IgM surrogate antigens tethered on the surface of PLB membranes by either H12-D-domain (red color) or streptavidin (blue color). Each dot represents a single measurement for the mFI of the tethered IgG surrogate antigens by Image J software. Bars represent means ± SD. Two-tailed t tests were performed for statistical comparisons. (B) Statistical quantification for the accumulation of human IgM-BCRs into the immunological synapse as measured by the mFI of BCR within the immunological synapse from Ramos human B cells that were placed on PLB membranes presenting the same amount of IgG surrogate antigen as shown in A that were tethered by either H12-D-domain or streptavidin. Each dot represents a single measurement for the mFI of IgM-BCRs by Image J software. Bars represent means ± SD. Two-tailed t tests were performed for statistical comparisons. (C) Statistical quantification for the accumulation of pSyk signaling molecules into the immunological synapse as measured by the mFI of BCR within the immunological synapse from Ramos human B cells that were placed on PLB membranes presenting the same amount of IgG surrogate antigen as shown in A that were tethered by either H12-D-domain or streptavidin. Each dot represents a single measurement for the mFI of pSyk by Image J software. Bars represent means ± SD. Two-tailed t tests were performed for statistical comparisons.

    Techniques Used: Fluorescence, Software, Two Tailed Test

    39) Product Images from "An Autoimmune Basis for Raynaud’s Phenomenon: Murine Model and Human Disease"

    Article Title: An Autoimmune Basis for Raynaud’s Phenomenon: Murine Model and Human Disease

    Journal: Arthritis & rheumatology (Hoboken, N.J.)

    doi: 10.1002/art.40505

    K10-dependent endothelial apoptosis. A . HUVEC were exposed for 24 hours to 10% dilutions of sera from healthy subjects (Ctrl) or RNP+ patients with or without Raynaud’s (RP+ or -) and anti-K10 IgG antibodies (ELISA > 2 S.D. above healthy subject mean to be anti-K10+), loaded with CellEvent Caspase 3/7 Green Detection Reagent (10μM/ml) and assayed for green absorbance in 24 hours in duplicate wells. Anti-K10+ sera induced high levels of caspase activity (an indicator of apoptosis) from patients with or without RP. ** t test p
    Figure Legend Snippet: K10-dependent endothelial apoptosis. A . HUVEC were exposed for 24 hours to 10% dilutions of sera from healthy subjects (Ctrl) or RNP+ patients with or without Raynaud’s (RP+ or -) and anti-K10 IgG antibodies (ELISA > 2 S.D. above healthy subject mean to be anti-K10+), loaded with CellEvent Caspase 3/7 Green Detection Reagent (10μM/ml) and assayed for green absorbance in 24 hours in duplicate wells. Anti-K10+ sera induced high levels of caspase activity (an indicator of apoptosis) from patients with or without RP. ** t test p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Activity Assay

    40) Product Images from "Immunological and Clinical Effect of Diet Modulation of the Gut Microbiome in Multiple Sclerosis Patients: A Pilot Study"

    Article Title: Immunological and Clinical Effect of Diet Modulation of the Gut Microbiome in Multiple Sclerosis Patients: A Pilot Study

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01391

    CD4+/IL-17+ and CD4+/PD-1+ T lymphocytes are decreased and CD14+/PD-L1+ cells are increased in multiple sclerosis (MS) patients following a high-vegetable/low-protein (HV/LP) diet. IL-17+/CD4+ T lymphocytes (A) ; PD-1+/CD4+T lymphocytes (B) ; and PD-L1+/CD14+ cells (C) . Representative results obtained in unstimulated peripheral blood mononuclear cell of MS patients who were following either a Western Diet (WD) or a HV/LP diet are shown. Top right corners show the percentage of CD4+/IL-17+, CD4+/PD-1+ T cells and of CD14+/PD-L1+ cells. Summary results are shown in (D–F) . The boxes stretch from the 25th to the 75th percentile; the lines across the boxes indicate the median values; the lines stretching from the boxes indicate extreme values. Statistical significance is shown. Blood samples were collected at enrollment, i.e., at least after 1 year of either WD or either HV/LP diet.
    Figure Legend Snippet: CD4+/IL-17+ and CD4+/PD-1+ T lymphocytes are decreased and CD14+/PD-L1+ cells are increased in multiple sclerosis (MS) patients following a high-vegetable/low-protein (HV/LP) diet. IL-17+/CD4+ T lymphocytes (A) ; PD-1+/CD4+T lymphocytes (B) ; and PD-L1+/CD14+ cells (C) . Representative results obtained in unstimulated peripheral blood mononuclear cell of MS patients who were following either a Western Diet (WD) or a HV/LP diet are shown. Top right corners show the percentage of CD4+/IL-17+, CD4+/PD-1+ T cells and of CD14+/PD-L1+ cells. Summary results are shown in (D–F) . The boxes stretch from the 25th to the 75th percentile; the lines across the boxes indicate the median values; the lines stretching from the boxes indicate extreme values. Statistical significance is shown. Blood samples were collected at enrollment, i.e., at least after 1 year of either WD or either HV/LP diet.

    Techniques Used: Mass Spectrometry, Western Blot

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  • 99
    Thermo Fisher anti human igg conjugate
    Rotavirus (RV) VP6‐specific antibodies and T cell responses. VP6‐specific immune responses were detected following immunization with 0·3 µg norovirus (NoV) GII.4 virus‐like particles (VLPs) together with VP6 nanotubes (VP6T) or nanospheres (VP6S). Control (Ctrl) mice received phosphate‐buffered saline (PBS) only. (a) Serum anti‐VP6 <t>IgG</t> of individual mice were tested in enzyme‐linked immunosorbent assay (ELISA) and the mean titration curves of each experimental group are shown. Bars represent log 10 geometric mean titres with 95% confidence intervals. For negative mice sera, an arbitrary titre of 1 : 100 (half the starting serum dilution, 1 : 200) was assigned. VP6‐specific IFN‐γ (b) and interleukin (IL)‐4 (c) production by T cells was tested stimulating the cells with VP6‐specific R6‐2 peptide, RV Wa and WC3 cell culture antigens, mock antigen or recombinant VP6 protein (rVP6). Results are expressed as the mean spot‐forming cells (SFC)/10 6 splenocytes of the duplicate wells with standard errors of the mean. The experiments were repeated two or more times with similar results.
    Anti Human Igg Conjugate, 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
    https://www.bioz.com/result/anti human igg conjugate/product/Thermo Fisher
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    anti human igg conjugate - by Bioz Stars, 2020-07
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    90
    Thermo Fisher texas red conjugated anti human igg antibody
    Immunohistochemical studies to detect deposition of aβ2GPI and <t>NHIgG</t> in placenta and fetal brain. Expression of β 2 GPI protein in placenta and fetal brain in control mice . aβ2GPI and NHIgG deposition in mouse tissue (placenta and fetal cortical brain) was detected using an antihuman <t>IgG</t> antibody labelled with Texas Red. A- detection of aβ 2 GPI in the placenta from mouse injected aβ 2 GPI. B- Detection of aβ 2 GPI in fetal cortical brain from a mouse injected with aβ 2 GPI. C- Detection of NHIgG in the placenta from a mouse injected with NHIgG. D- Detection of NHIgG in the fetal cortical brain in a mouse injected with NHIgG. Expression of β 2 GPI in placenta (E) and fetal brain (F) was detected with FITC-conjugated antibodies (green fluorescence). Diamidino-2-phenylindole (DAPI) was used for nuclear counterstains in all immunofluorescence studies. Data are representative of observations in 5–6 mice per group. 10 views per slide were analyzed in each experimental condition.
    Texas Red Conjugated Anti Human Igg Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/texas red conjugated anti human igg antibody/product/Thermo Fisher
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    93
    Thermo Fisher antibody mouse anti human igg2
    Box plot analysis of antibody reactivity to recombinant antigens rPstS-1. Recombinant proteins (from E. coli and P. pastoris ) were tested with PPD-negative (n = 5), PPD-positive sera (n = 5) at 1:100 and active-TB sera (n = 30) at 1:500 dilution. The reactivity is reported as difference of the absorbance units observed for the different group of sera by ELISA using a <t>IgG2</t> conjugated with AP. Different sera of each group were tested by duplicate. Mean (dashed lane), median, 10th, 25th, 75th and 90th percentiles as vertical boxes with error bars are shown. Dunn´s test ws used for all pairwise comparisons and comparisons against a control group (PBS) following rank-based ANOVA
    Antibody Mouse Anti Human Igg2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Rotavirus (RV) VP6‐specific antibodies and T cell responses. VP6‐specific immune responses were detected following immunization with 0·3 µg norovirus (NoV) GII.4 virus‐like particles (VLPs) together with VP6 nanotubes (VP6T) or nanospheres (VP6S). Control (Ctrl) mice received phosphate‐buffered saline (PBS) only. (a) Serum anti‐VP6 IgG of individual mice were tested in enzyme‐linked immunosorbent assay (ELISA) and the mean titration curves of each experimental group are shown. Bars represent log 10 geometric mean titres with 95% confidence intervals. For negative mice sera, an arbitrary titre of 1 : 100 (half the starting serum dilution, 1 : 200) was assigned. VP6‐specific IFN‐γ (b) and interleukin (IL)‐4 (c) production by T cells was tested stimulating the cells with VP6‐specific R6‐2 peptide, RV Wa and WC3 cell culture antigens, mock antigen or recombinant VP6 protein (rVP6). Results are expressed as the mean spot‐forming cells (SFC)/10 6 splenocytes of the duplicate wells with standard errors of the mean. The experiments were repeated two or more times with similar results.

    Journal: Clinical and Experimental Immunology

    Article Title: Rotavirus capsid VP6 tubular and spherical nanostructures act as local adjuvants when co‐delivered with norovirus VLPs

    doi: 10.1111/cei.12977

    Figure Lengend Snippet: Rotavirus (RV) VP6‐specific antibodies and T cell responses. VP6‐specific immune responses were detected following immunization with 0·3 µg norovirus (NoV) GII.4 virus‐like particles (VLPs) together with VP6 nanotubes (VP6T) or nanospheres (VP6S). Control (Ctrl) mice received phosphate‐buffered saline (PBS) only. (a) Serum anti‐VP6 IgG of individual mice were tested in enzyme‐linked immunosorbent assay (ELISA) and the mean titration curves of each experimental group are shown. Bars represent log 10 geometric mean titres with 95% confidence intervals. For negative mice sera, an arbitrary titre of 1 : 100 (half the starting serum dilution, 1 : 200) was assigned. VP6‐specific IFN‐γ (b) and interleukin (IL)‐4 (c) production by T cells was tested stimulating the cells with VP6‐specific R6‐2 peptide, RV Wa and WC3 cell culture antigens, mock antigen or recombinant VP6 protein (rVP6). Results are expressed as the mean spot‐forming cells (SFC)/10 6 splenocytes of the duplicate wells with standard errors of the mean. The experiments were repeated two or more times with similar results.

    Article Snippet: The bound VLPs were detected using human anti‐NoV detection serum and anti‐human IgG conjugate (Novex; Thermo Fisher Scientific, Fremont, CA, USA).

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay, Titration, Cell Culture, Recombinant

    Immunohistochemical studies to detect deposition of aβ2GPI and NHIgG in placenta and fetal brain. Expression of β 2 GPI protein in placenta and fetal brain in control mice . aβ2GPI and NHIgG deposition in mouse tissue (placenta and fetal cortical brain) was detected using an antihuman IgG antibody labelled with Texas Red. A- detection of aβ 2 GPI in the placenta from mouse injected aβ 2 GPI. B- Detection of aβ 2 GPI in fetal cortical brain from a mouse injected with aβ 2 GPI. C- Detection of NHIgG in the placenta from a mouse injected with NHIgG. D- Detection of NHIgG in the fetal cortical brain in a mouse injected with NHIgG. Expression of β 2 GPI in placenta (E) and fetal brain (F) was detected with FITC-conjugated antibodies (green fluorescence). Diamidino-2-phenylindole (DAPI) was used for nuclear counterstains in all immunofluorescence studies. Data are representative of observations in 5–6 mice per group. 10 views per slide were analyzed in each experimental condition.

    Journal: Journal of autoimmunity

    Article Title: Complement inhibition by hydroxychloroquine prevents placental and fetal brain abnormalities in antiphospholipid syndrome

    doi: 10.1016/j.jaut.2016.04.008

    Figure Lengend Snippet: Immunohistochemical studies to detect deposition of aβ2GPI and NHIgG in placenta and fetal brain. Expression of β 2 GPI protein in placenta and fetal brain in control mice . aβ2GPI and NHIgG deposition in mouse tissue (placenta and fetal cortical brain) was detected using an antihuman IgG antibody labelled with Texas Red. A- detection of aβ 2 GPI in the placenta from mouse injected aβ 2 GPI. B- Detection of aβ 2 GPI in fetal cortical brain from a mouse injected with aβ 2 GPI. C- Detection of NHIgG in the placenta from a mouse injected with NHIgG. D- Detection of NHIgG in the fetal cortical brain in a mouse injected with NHIgG. Expression of β 2 GPI in placenta (E) and fetal brain (F) was detected with FITC-conjugated antibodies (green fluorescence). Diamidino-2-phenylindole (DAPI) was used for nuclear counterstains in all immunofluorescence studies. Data are representative of observations in 5–6 mice per group. 10 views per slide were analyzed in each experimental condition.

    Article Snippet: Texas Red-conjugated anti human IgG antibody was also used to visualize NHIgG in placenta and fetal brain in NHIgG-treated mice.

    Techniques: Immunohistochemistry, Expressing, Mouse Assay, Injection, Fluorescence, Immunofluorescence

    Box plot analysis of antibody reactivity to recombinant antigens rPstS-1. Recombinant proteins (from E. coli and P. pastoris ) were tested with PPD-negative (n = 5), PPD-positive sera (n = 5) at 1:100 and active-TB sera (n = 30) at 1:500 dilution. The reactivity is reported as difference of the absorbance units observed for the different group of sera by ELISA using a IgG2 conjugated with AP. Different sera of each group were tested by duplicate. Mean (dashed lane), median, 10th, 25th, 75th and 90th percentiles as vertical boxes with error bars are shown. Dunn´s test ws used for all pairwise comparisons and comparisons against a control group (PBS) following rank-based ANOVA

    Journal: Microbial Cell Factories

    Article Title: Recombinant O-mannosylated protein production (PstS-1) from Mycobacterium tuberculosis in Pichia pastoris (Komagataella phaffii) as a tool to study tuberculosis infection

    doi: 10.1186/s12934-019-1059-3

    Figure Lengend Snippet: Box plot analysis of antibody reactivity to recombinant antigens rPstS-1. Recombinant proteins (from E. coli and P. pastoris ) were tested with PPD-negative (n = 5), PPD-positive sera (n = 5) at 1:100 and active-TB sera (n = 30) at 1:500 dilution. The reactivity is reported as difference of the absorbance units observed for the different group of sera by ELISA using a IgG2 conjugated with AP. Different sera of each group were tested by duplicate. Mean (dashed lane), median, 10th, 25th, 75th and 90th percentiles as vertical boxes with error bars are shown. Dunn´s test ws used for all pairwise comparisons and comparisons against a control group (PBS) following rank-based ANOVA

    Article Snippet: The wells were then incubated with 100 μL of secondary antibody mouse anti-human IgG2 (1:500 PBS/Tween 0.05%) coupled to alkaline phosphatase (Thermo Fisher Scientific), for 1.5 h at 37 °C in the dark and washed four times with 250 μL of Tween 20 (0.05%) in PBS.

    Techniques: Recombinant, Enzyme-linked Immunosorbent Assay