goat anti mouse igm heavy chain cross adsorbed secondary antibody  (Thermo Fisher)


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    Name:
    Goat anti Mouse IgM Heavy Chain Cross Adsorbed Secondary Antibody
    Description:
    Goat anti Mouse IgM Heavy Chain Cross Adsorbed Secondary Antibody for IF ICC IHC
    Catalog Number:
    a21042
    Price:
    None
    Applications:
    Antibodies and Secondary Detection|Cell Analysis|Secondary Detection
    Category:
    Antibodies Secondary Detection Reagents
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    Structured Review

    Thermo Fisher goat anti mouse igm heavy chain cross adsorbed secondary antibody
    States of inflammation and fibrosis in the liver in the absence of serum <t>IgM-free</t> <t>AIM.</t> ( a ) The mRNA levels of various genes responsive to different types of stresses (i.e., endoplasmic reticulum, mitochondrial, or oxidative stress) addressed by qPCR using RNA from the whole liver of AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 5–6 for 12-week HFD per group). Error bar indicates the SEM. (b,c) The mRNA levels of inflammatory cytokine genes in the liver, as assessed by qPCR using the RNA as in ( a ). (d ) Representative photomicrographs of liver from AIM-felinized, WT, AIM −/− , and Δsμ mice fed an HFD for 12 weeks stained with Sirius red Stain. Scale bars, 100 μm. (e) The mRNA levels of fibrogenetic genes in the liver, as assessed by qPCR using the same RNA as in ( a ). In (a – c) and (e) , *is used to represent the statistical significance between the values of each mouse strain group within the same period, whereas # is attached to the bar of 12 w when the value of 12 w was significantly changed compared with that of 0 w in the same mouse strain group.
    Goat anti Mouse IgM Heavy Chain Cross Adsorbed Secondary Antibody for IF ICC IHC
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    Images

    1) Product Images from "Independent modes of disease repair by AIM protein distinguished in AIM-felinized mice"

    Article Title: Independent modes of disease repair by AIM protein distinguished in AIM-felinized mice

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31580-6

    States of inflammation and fibrosis in the liver in the absence of serum IgM-free AIM. ( a ) The mRNA levels of various genes responsive to different types of stresses (i.e., endoplasmic reticulum, mitochondrial, or oxidative stress) addressed by qPCR using RNA from the whole liver of AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 5–6 for 12-week HFD per group). Error bar indicates the SEM. (b,c) The mRNA levels of inflammatory cytokine genes in the liver, as assessed by qPCR using the RNA as in ( a ). (d ) Representative photomicrographs of liver from AIM-felinized, WT, AIM −/− , and Δsμ mice fed an HFD for 12 weeks stained with Sirius red Stain. Scale bars, 100 μm. (e) The mRNA levels of fibrogenetic genes in the liver, as assessed by qPCR using the same RNA as in ( a ). In (a – c) and (e) , *is used to represent the statistical significance between the values of each mouse strain group within the same period, whereas # is attached to the bar of 12 w when the value of 12 w was significantly changed compared with that of 0 w in the same mouse strain group.
    Figure Legend Snippet: States of inflammation and fibrosis in the liver in the absence of serum IgM-free AIM. ( a ) The mRNA levels of various genes responsive to different types of stresses (i.e., endoplasmic reticulum, mitochondrial, or oxidative stress) addressed by qPCR using RNA from the whole liver of AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 5–6 for 12-week HFD per group). Error bar indicates the SEM. (b,c) The mRNA levels of inflammatory cytokine genes in the liver, as assessed by qPCR using the RNA as in ( a ). (d ) Representative photomicrographs of liver from AIM-felinized, WT, AIM −/− , and Δsμ mice fed an HFD for 12 weeks stained with Sirius red Stain. Scale bars, 100 μm. (e) The mRNA levels of fibrogenetic genes in the liver, as assessed by qPCR using the same RNA as in ( a ). In (a – c) and (e) , *is used to represent the statistical significance between the values of each mouse strain group within the same period, whereas # is attached to the bar of 12 w when the value of 12 w was significantly changed compared with that of 0 w in the same mouse strain group.

    Techniques Used: Real-time Polymerase Chain Reaction, Mouse Assay, Staining

    Effect of serum IgM-free AIM on obesity and liver steatosis. ( a,b) Weights from AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 6–9 for 12-week HFD per group). Error bar indicates the SEM. (a) Body weights. (b ) Weights of epidydimal adipose tissues. (c) Representative photomicrographs of epididymal fat tissues from AIM-felinized, WT, AIM −/− , and Δsµ mice fed an HFD for 12 weeks stained with H E. Adipocyte sizes of 50 independent adipocytes in different areas were evaluated. Results are presented as average ± SEM (in μm 2 ). Scale bars, 100 μm. ( d ) Representative photomicrographs of epididymal fat tissues from AIM-felinized, WT, and AIM −/− mice (fed an HFD for 12 weeks) stained for AIM (blue), F4/80 (macrophage marker; green), and IgM (red, WT and AIM −/− mice. See Supplementary Fig. 1 for AIM-felinized mice). Yellow arrows represent where IgM-free AIM signals exist, while red arrows indicate where AIM is co-stained with IgM. Scale bars, 100 μm. (e) The mRNA levels of AIM and F4/80 were assessed by qPCR using RNA isolated from epididymal fat in WT mice before or after being fed an HFD for 12 weeks. Values were normalized to those of GAPDH and presented as the expression relative to that of AIM from lean WT mice liver and of F4/80 from fat tissues before being fed an HFD (n = 4 per group). Error bar indicates the SEM. (f) Representative photomicrographs of liver from AIM-felinized, WT, AIM −/− , and Δsµ mice fed an HFD for 12 weeks stained with H E. Scale bars, 100 µm. (g) Liver weights and TG contents from AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 6–9 for 12-week HFD per group). Error bar indicates the SEM.
    Figure Legend Snippet: Effect of serum IgM-free AIM on obesity and liver steatosis. ( a,b) Weights from AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 6–9 for 12-week HFD per group). Error bar indicates the SEM. (a) Body weights. (b ) Weights of epidydimal adipose tissues. (c) Representative photomicrographs of epididymal fat tissues from AIM-felinized, WT, AIM −/− , and Δsµ mice fed an HFD for 12 weeks stained with H E. Adipocyte sizes of 50 independent adipocytes in different areas were evaluated. Results are presented as average ± SEM (in μm 2 ). Scale bars, 100 μm. ( d ) Representative photomicrographs of epididymal fat tissues from AIM-felinized, WT, and AIM −/− mice (fed an HFD for 12 weeks) stained for AIM (blue), F4/80 (macrophage marker; green), and IgM (red, WT and AIM −/− mice. See Supplementary Fig. 1 for AIM-felinized mice). Yellow arrows represent where IgM-free AIM signals exist, while red arrows indicate where AIM is co-stained with IgM. Scale bars, 100 μm. (e) The mRNA levels of AIM and F4/80 were assessed by qPCR using RNA isolated from epididymal fat in WT mice before or after being fed an HFD for 12 weeks. Values were normalized to those of GAPDH and presented as the expression relative to that of AIM from lean WT mice liver and of F4/80 from fat tissues before being fed an HFD (n = 4 per group). Error bar indicates the SEM. (f) Representative photomicrographs of liver from AIM-felinized, WT, AIM −/− , and Δsµ mice fed an HFD for 12 weeks stained with H E. Scale bars, 100 µm. (g) Liver weights and TG contents from AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 6–9 for 12-week HFD per group). Error bar indicates the SEM.

    Techniques Used: Mouse Assay, Staining, Marker, Real-time Polymerase Chain Reaction, Isolation, Expressing

    2) Product Images from "Suppression of Glomerulonephritis in NZB/NZW Lupus Prone Mice by Adoptive Transfer of Ex Vivo Expanded Regulatory T Cells"

    Article Title: Suppression of Glomerulonephritis in NZB/NZW Lupus Prone Mice by Adoptive Transfer of Ex Vivo Expanded Regulatory T Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0006031

    Inhibition of immune complex renal damage in T reg treated mice. Typical kidney sections demonstrating less severe IgG and IgM deposition in mice receiving adoptive T reg transfer ( Figure 6A–B respectively) as compared to mice receiving either control therapy (T eff control mouse, Figure 6C–D ).
    Figure Legend Snippet: Inhibition of immune complex renal damage in T reg treated mice. Typical kidney sections demonstrating less severe IgG and IgM deposition in mice receiving adoptive T reg transfer ( Figure 6A–B respectively) as compared to mice receiving either control therapy (T eff control mouse, Figure 6C–D ).

    Techniques Used: Inhibition, Mouse Assay

    3) Product Images from "A public antibody lineage that potently inhibits malaria infection by dual binding to the circumsporozoite protein"

    Article Title: A public antibody lineage that potently inhibits malaria infection by dual binding to the circumsporozoite protein

    Journal: Nature medicine

    doi: 10.1038/nm.4513

    Immunization with PfSPZ Vaccine induces robust antibody responses in malaria-exposed individuals a, Protocol of PfSPZ immunization of Tanzanian adults. b, Binding of serum IgM and IgG antibodies to PfSPZ. Median fluorescence intensity (MFI) values are for binding at a 1/1000 serum dilution (representative of n=2 independent experiments). Samples in red, black and blue are from protected (U, G, V, H), non-protected (NP) and placebo (C) volunteers, respectively. Results for donor W are not shown as this donor was immunized with a lower dose of PfSPZ. c, Staining of PfSPZ by serum from a European blood donor, serum from a protected individual (donor H) and a monoclonal antibody (MGU8) (representative of n=3 independent experiments). d, Dose-dependent binding of three representative antibodies to PfSPZ measured by flow cytometry (representative of n=2 independent experiments). e, Binding values of the panel of IgG monoclonal antibodies to PfSPZ (representative of n=2 independent experiments). The values indicate the concentration of antibody required to reach a 10,000 MFI. f, Number of PfSPZ-binding IgG and IgM monoclonal antibodies isolated from protected donors G and U. g, Number of mutations in the heavy chains of IgG (n=19 antibodies) and IgM (n=65 antibodies) isolated from the Tanzanian volunteers. These values were calculated by adding the number of VH and JH mutations. Results are shown as mean ± s.d.. A two-sided t-test was used to compare the number of mutations.
    Figure Legend Snippet: Immunization with PfSPZ Vaccine induces robust antibody responses in malaria-exposed individuals a, Protocol of PfSPZ immunization of Tanzanian adults. b, Binding of serum IgM and IgG antibodies to PfSPZ. Median fluorescence intensity (MFI) values are for binding at a 1/1000 serum dilution (representative of n=2 independent experiments). Samples in red, black and blue are from protected (U, G, V, H), non-protected (NP) and placebo (C) volunteers, respectively. Results for donor W are not shown as this donor was immunized with a lower dose of PfSPZ. c, Staining of PfSPZ by serum from a European blood donor, serum from a protected individual (donor H) and a monoclonal antibody (MGU8) (representative of n=3 independent experiments). d, Dose-dependent binding of three representative antibodies to PfSPZ measured by flow cytometry (representative of n=2 independent experiments). e, Binding values of the panel of IgG monoclonal antibodies to PfSPZ (representative of n=2 independent experiments). The values indicate the concentration of antibody required to reach a 10,000 MFI. f, Number of PfSPZ-binding IgG and IgM monoclonal antibodies isolated from protected donors G and U. g, Number of mutations in the heavy chains of IgG (n=19 antibodies) and IgM (n=65 antibodies) isolated from the Tanzanian volunteers. These values were calculated by adding the number of VH and JH mutations. Results are shown as mean ± s.d.. A two-sided t-test was used to compare the number of mutations.

    Techniques Used: Binding Assay, Fluorescence, Staining, Flow Cytometry, Cytometry, Concentration Assay, Isolation

    4) Product Images from "CXCL12 induces migration of oligodendrocyte precursor cells through the CXCR4-activated MEK/ERK and PI3K/AKT pathways"

    Article Title: CXCL12 induces migration of oligodendrocyte precursor cells through the CXCR4-activated MEK/ERK and PI3K/AKT pathways

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2018.9444

    Isolation and identification of OPCs. (A) Immunostaining demonstrated that OPCs were positive for NG2. (B) Immunostaining demonstrated that OPCs were positive for PDGFR-α. (C) Immunostaining showed that the differentiated OPCs were positive for O4. (D) Immunostaining showed that the differentiated OPCs were positive for MBP. Nuclei were stained with DAPI (blue). NG2, neural/glial antigen 2; PDGFR-α, platelet-derived growth factor receptor-α; OPCs, oligodendrocyte precursor cells; O4, oligodendrocyte marker O4; MBP, myelin basic protein.
    Figure Legend Snippet: Isolation and identification of OPCs. (A) Immunostaining demonstrated that OPCs were positive for NG2. (B) Immunostaining demonstrated that OPCs were positive for PDGFR-α. (C) Immunostaining showed that the differentiated OPCs were positive for O4. (D) Immunostaining showed that the differentiated OPCs were positive for MBP. Nuclei were stained with DAPI (blue). NG2, neural/glial antigen 2; PDGFR-α, platelet-derived growth factor receptor-α; OPCs, oligodendrocyte precursor cells; O4, oligodendrocyte marker O4; MBP, myelin basic protein.

    Techniques Used: Isolation, Immunostaining, Staining, Derivative Assay, Marker

    5) Product Images from "Heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs) function as endocytic receptors for an internalizing anti-nucleic acid antibody"

    Article Title: Heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs) function as endocytic receptors for an internalizing anti-nucleic acid antibody

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-14793-z

    3D8 scFv colocalizes intracellularly with proteoglycans and caveolin-1 upon binding to cell surface proteoglycans (HSPGs and CSPGs). ( a,b ) Confocal microscopy to detect intracellular colocalization of 3D8 scFv, proteoglycans (HSPGs and CSPGs), and caveolin-1. HeLa cells were incubated with 3D8 scFv (10 μM) for 1 h, 6 h, or 12 h at 37 °C. After fixation and permeabilization, cells were incubated at 4 °C overnight with a primary antibody mixture comprising rabbit (IgG) anti-3D8 scFv, mouse (IgG) anti-caveolin-1, and mouse (IgM) anti-HS antibodies ( a ), or a mixture of rabbit anti-3D8 scFv, mouse anti-caveolin-1, and mouse (IgM) anti-CS antibodies ( b ). Thereafter, cells were incubated with a mixture of Alexa Fluor 647-conjugated goat anti-rabbit IgG, TRITC-conjugated goat anti-mouse IgG, and Alexa Fluor 488-conjugated goat anti-mouse IgM/μ chain-specific antibody. Enlarged images of the boxed areas in the upper panel are shown in the lower panel. ( c,d ) HeLa cells were incubated for 6 h at 37 °C with HW6, followed rabbit anti-His tag and Alexa Fluor 647-conjugated goat anti-rabbit IgG antibodies. Bar , 10 μm.
    Figure Legend Snippet: 3D8 scFv colocalizes intracellularly with proteoglycans and caveolin-1 upon binding to cell surface proteoglycans (HSPGs and CSPGs). ( a,b ) Confocal microscopy to detect intracellular colocalization of 3D8 scFv, proteoglycans (HSPGs and CSPGs), and caveolin-1. HeLa cells were incubated with 3D8 scFv (10 μM) for 1 h, 6 h, or 12 h at 37 °C. After fixation and permeabilization, cells were incubated at 4 °C overnight with a primary antibody mixture comprising rabbit (IgG) anti-3D8 scFv, mouse (IgG) anti-caveolin-1, and mouse (IgM) anti-HS antibodies ( a ), or a mixture of rabbit anti-3D8 scFv, mouse anti-caveolin-1, and mouse (IgM) anti-CS antibodies ( b ). Thereafter, cells were incubated with a mixture of Alexa Fluor 647-conjugated goat anti-rabbit IgG, TRITC-conjugated goat anti-mouse IgG, and Alexa Fluor 488-conjugated goat anti-mouse IgM/μ chain-specific antibody. Enlarged images of the boxed areas in the upper panel are shown in the lower panel. ( c,d ) HeLa cells were incubated for 6 h at 37 °C with HW6, followed rabbit anti-His tag and Alexa Fluor 647-conjugated goat anti-rabbit IgG antibodies. Bar , 10 μm.

    Techniques Used: Binding Assay, Confocal Microscopy, Incubation

    3D8 scFv binds to cell surface HSPGs and CSPGs. ( a ) Flow cytometry analysis of cell surface expression of endogenous HSPGs and CSPGs in HeLa cells. ( b ) Confocal microscopy to detect 3D8 scFv binding to cell surface HSPGs and CSPGs. HeLa cells were incubated for 1 h at 4 °C with 3D8 scFv (10 μM) and HW6 scFv (10 μM) (negative control). Thereafter, cells were incubated with a primary antibody mixture containing a rabbit anti-3D8 scFv antibody and a mouse IgM anti-HS antibody, or a rabbit anti-3D8 scFv antibody and a mouse IgM anti-CS antibody. After washing, cells were incubated with a secondary antibody mixture comprising TRITC-conjugated anti-rabbit IgG and Alexa Fluor 488-conjugated anti-mouse IgM. ( c ) Confocal microscopy to detect 3D8 scFv binding to cell surface HSPGs in the presence of soluble HS and CS chains. HeLa cells were incubated for 1 h at 4 °C with 3D8 scFv (10 μM) in the absence ( upper panel ) or presence ( middle or lower panel ) of heparin (10 μg/ml) or CS-A (10 μg/ml), followed by the procedures described in ( b ). ( d,e ) Flow cytometry ( d ) and confocal microscopy ( e ) to detect the cell surface sugar chains (HS and CS) and cell surface binding of 3D8 scFv to HeLa cells pre-treated with heparinase III (10 mIU/ml) and chondroitinase ABC (100 mIU/ml). ( b,c,e ) Nuclei were stained with Hoechst 33342 (blue). Bar , 10 μm.
    Figure Legend Snippet: 3D8 scFv binds to cell surface HSPGs and CSPGs. ( a ) Flow cytometry analysis of cell surface expression of endogenous HSPGs and CSPGs in HeLa cells. ( b ) Confocal microscopy to detect 3D8 scFv binding to cell surface HSPGs and CSPGs. HeLa cells were incubated for 1 h at 4 °C with 3D8 scFv (10 μM) and HW6 scFv (10 μM) (negative control). Thereafter, cells were incubated with a primary antibody mixture containing a rabbit anti-3D8 scFv antibody and a mouse IgM anti-HS antibody, or a rabbit anti-3D8 scFv antibody and a mouse IgM anti-CS antibody. After washing, cells were incubated with a secondary antibody mixture comprising TRITC-conjugated anti-rabbit IgG and Alexa Fluor 488-conjugated anti-mouse IgM. ( c ) Confocal microscopy to detect 3D8 scFv binding to cell surface HSPGs in the presence of soluble HS and CS chains. HeLa cells were incubated for 1 h at 4 °C with 3D8 scFv (10 μM) in the absence ( upper panel ) or presence ( middle or lower panel ) of heparin (10 μg/ml) or CS-A (10 μg/ml), followed by the procedures described in ( b ). ( d,e ) Flow cytometry ( d ) and confocal microscopy ( e ) to detect the cell surface sugar chains (HS and CS) and cell surface binding of 3D8 scFv to HeLa cells pre-treated with heparinase III (10 mIU/ml) and chondroitinase ABC (100 mIU/ml). ( b,c,e ) Nuclei were stained with Hoechst 33342 (blue). Bar , 10 μm.

    Techniques Used: Flow Cytometry, Cytometry, Expressing, Confocal Microscopy, Binding Assay, Incubation, Negative Control, Staining

    6) Product Images from "MOG cell-based assay detects non-MS patients with inflammatory neurologic disease"

    Article Title: MOG cell-based assay detects non-MS patients with inflammatory neurologic disease

    Journal: Neurology® Neuroimmunology & Neuroinflammation

    doi: 10.1212/NXI.0000000000000089

    Antibodies to MOG using different secondary antibodies: Anti-human IgG (H + L), IgG1, or IgM (A) Comparison of binding to full-length myelin oligodendrocyte glycoprotein (FL-MOG) using anti-human IgG (H + L), anti-IgM, or anti-IgG1 secondary antibodies with 3 different test sera (a-c) and a healthy control serum (con). (B) IgM and (C) IgG1 binding scores for patients and healthy controls (HC). (D.a) PIRES2-DsRed2-FL-MOG transiently transfected HEK cells are separated into cells that express MOG and DsRed2 well (in the upper section of the graph) or poorly or not at all (lowest section of the graph). (D.b) Healthy control sera (upper panels) causes a specific shift in the MOG-transfected cells compared to the untransfected cells when anti-human IgG (H + L) or anti-human IgM secondary antibodies are used (arrows), but not when anti-human IgG1 secondary antibodies are used. The lower panels show higher shifts in sera positive for FL-MOG antibodies compared to controls in the upper panel. (E) Fifteen samples that were IgG (H + L) positive and 5 healthy controls were tested on flow cytometry with anti-IgM or IgG1. A high cutoff is generated with anti-human IgM secondary antibody (ΔMFI of 270) vs a ΔMFI of 2.5 for the anti-human IgG1 antibody. Of note, one IgM-positive patient is IgG1 negative (blue circle). Ab = antibody; AQP4 = aquaporin-4; CBA = cell-based assay; MFI = mean fluorescence intensity; MS = multiple sclerosis.
    Figure Legend Snippet: Antibodies to MOG using different secondary antibodies: Anti-human IgG (H + L), IgG1, or IgM (A) Comparison of binding to full-length myelin oligodendrocyte glycoprotein (FL-MOG) using anti-human IgG (H + L), anti-IgM, or anti-IgG1 secondary antibodies with 3 different test sera (a-c) and a healthy control serum (con). (B) IgM and (C) IgG1 binding scores for patients and healthy controls (HC). (D.a) PIRES2-DsRed2-FL-MOG transiently transfected HEK cells are separated into cells that express MOG and DsRed2 well (in the upper section of the graph) or poorly or not at all (lowest section of the graph). (D.b) Healthy control sera (upper panels) causes a specific shift in the MOG-transfected cells compared to the untransfected cells when anti-human IgG (H + L) or anti-human IgM secondary antibodies are used (arrows), but not when anti-human IgG1 secondary antibodies are used. The lower panels show higher shifts in sera positive for FL-MOG antibodies compared to controls in the upper panel. (E) Fifteen samples that were IgG (H + L) positive and 5 healthy controls were tested on flow cytometry with anti-IgM or IgG1. A high cutoff is generated with anti-human IgM secondary antibody (ΔMFI of 270) vs a ΔMFI of 2.5 for the anti-human IgG1 antibody. Of note, one IgM-positive patient is IgG1 negative (blue circle). Ab = antibody; AQP4 = aquaporin-4; CBA = cell-based assay; MFI = mean fluorescence intensity; MS = multiple sclerosis.

    Techniques Used: Binding Assay, Transfection, Flow Cytometry, Cytometry, Generated, Crocin Bleaching Assay, Cell Based Assay, Fluorescence, Mass Spectrometry

    7) Product Images from "Heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs) function as endocytic receptors for an internalizing anti-nucleic acid antibody"

    Article Title: Heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs) function as endocytic receptors for an internalizing anti-nucleic acid antibody

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-14793-z

    3D8 scFv colocalizes intracellularly with proteoglycans and caveolin-1 upon binding to cell surface proteoglycans (HSPGs and CSPGs). ( a,b ) Confocal microscopy to detect intracellular colocalization of 3D8 scFv, proteoglycans (HSPGs and CSPGs), and caveolin-1. HeLa cells were incubated with 3D8 scFv (10 μM) for 1 h, 6 h, or 12 h at 37 °C. After fixation and permeabilization, cells were incubated at 4 °C overnight with a primary antibody mixture comprising rabbit (IgG) anti-3D8 scFv, mouse (IgG) anti-caveolin-1, and mouse (IgM) anti-HS antibodies ( a ), or a mixture of rabbit anti-3D8 scFv, mouse anti-caveolin-1, and mouse (IgM) anti-CS antibodies ( b ). Thereafter, cells were incubated with a mixture of Alexa Fluor 647-conjugated goat anti-rabbit IgG, TRITC-conjugated goat anti-mouse IgG, and Alexa Fluor 488-conjugated goat anti-mouse IgM/μ chain-specific antibody. Enlarged images of the boxed areas in the upper panel are shown in the lower panel. ( c,d ) HeLa cells were incubated for 6 h at 37 °C with HW6, followed rabbit anti-His tag and Alexa Fluor 647-conjugated goat anti-rabbit IgG antibodies. Bar , 10 μm.
    Figure Legend Snippet: 3D8 scFv colocalizes intracellularly with proteoglycans and caveolin-1 upon binding to cell surface proteoglycans (HSPGs and CSPGs). ( a,b ) Confocal microscopy to detect intracellular colocalization of 3D8 scFv, proteoglycans (HSPGs and CSPGs), and caveolin-1. HeLa cells were incubated with 3D8 scFv (10 μM) for 1 h, 6 h, or 12 h at 37 °C. After fixation and permeabilization, cells were incubated at 4 °C overnight with a primary antibody mixture comprising rabbit (IgG) anti-3D8 scFv, mouse (IgG) anti-caveolin-1, and mouse (IgM) anti-HS antibodies ( a ), or a mixture of rabbit anti-3D8 scFv, mouse anti-caveolin-1, and mouse (IgM) anti-CS antibodies ( b ). Thereafter, cells were incubated with a mixture of Alexa Fluor 647-conjugated goat anti-rabbit IgG, TRITC-conjugated goat anti-mouse IgG, and Alexa Fluor 488-conjugated goat anti-mouse IgM/μ chain-specific antibody. Enlarged images of the boxed areas in the upper panel are shown in the lower panel. ( c,d ) HeLa cells were incubated for 6 h at 37 °C with HW6, followed rabbit anti-His tag and Alexa Fluor 647-conjugated goat anti-rabbit IgG antibodies. Bar , 10 μm.

    Techniques Used: Binding Assay, Confocal Microscopy, Incubation

    3D8 scFv binds to cell surface HSPGs and CSPGs. ( a ) Flow cytometry analysis of cell surface expression of endogenous HSPGs and CSPGs in HeLa cells. ( b ) Confocal microscopy to detect 3D8 scFv binding to cell surface HSPGs and CSPGs. HeLa cells were incubated for 1 h at 4 °C with 3D8 scFv (10 μM) and HW6 scFv (10 μM) (negative control). Thereafter, cells were incubated with a primary antibody mixture containing a rabbit anti-3D8 scFv antibody and a mouse IgM anti-HS antibody, or a rabbit anti-3D8 scFv antibody and a mouse IgM anti-CS antibody. After washing, cells were incubated with a secondary antibody mixture comprising TRITC-conjugated anti-rabbit IgG and Alexa Fluor 488-conjugated anti-mouse IgM. ( c ) Confocal microscopy to detect 3D8 scFv binding to cell surface HSPGs in the presence of soluble HS and CS chains. HeLa cells were incubated for 1 h at 4 °C with 3D8 scFv (10 μM) in the absence ( upper panel ) or presence ( middle or lower panel ) of heparin (10 μg/ml) or CS-A (10 μg/ml), followed by the procedures described in ( b ). ( d,e ) Flow cytometry ( d ) and confocal microscopy ( e ) to detect the cell surface sugar chains (HS and CS) and cell surface binding of 3D8 scFv to HeLa cells pre-treated with heparinase III (10 mIU/ml) and chondroitinase ABC (100 mIU/ml). ( b,c,e ) Nuclei were stained with Hoechst 33342 (blue). Bar , 10 μm.
    Figure Legend Snippet: 3D8 scFv binds to cell surface HSPGs and CSPGs. ( a ) Flow cytometry analysis of cell surface expression of endogenous HSPGs and CSPGs in HeLa cells. ( b ) Confocal microscopy to detect 3D8 scFv binding to cell surface HSPGs and CSPGs. HeLa cells were incubated for 1 h at 4 °C with 3D8 scFv (10 μM) and HW6 scFv (10 μM) (negative control). Thereafter, cells were incubated with a primary antibody mixture containing a rabbit anti-3D8 scFv antibody and a mouse IgM anti-HS antibody, or a rabbit anti-3D8 scFv antibody and a mouse IgM anti-CS antibody. After washing, cells were incubated with a secondary antibody mixture comprising TRITC-conjugated anti-rabbit IgG and Alexa Fluor 488-conjugated anti-mouse IgM. ( c ) Confocal microscopy to detect 3D8 scFv binding to cell surface HSPGs in the presence of soluble HS and CS chains. HeLa cells were incubated for 1 h at 4 °C with 3D8 scFv (10 μM) in the absence ( upper panel ) or presence ( middle or lower panel ) of heparin (10 μg/ml) or CS-A (10 μg/ml), followed by the procedures described in ( b ). ( d,e ) Flow cytometry ( d ) and confocal microscopy ( e ) to detect the cell surface sugar chains (HS and CS) and cell surface binding of 3D8 scFv to HeLa cells pre-treated with heparinase III (10 mIU/ml) and chondroitinase ABC (100 mIU/ml). ( b,c,e ) Nuclei were stained with Hoechst 33342 (blue). Bar , 10 μm.

    Techniques Used: Flow Cytometry, Cytometry, Expressing, Confocal Microscopy, Binding Assay, Incubation, Negative Control, Staining

    8) Product Images from "CXCL12 induces migration of oligodendrocyte precursor cells through the CXCR4-activated MEK/ERK and PI3K/AKT pathways"

    Article Title: CXCL12 induces migration of oligodendrocyte precursor cells through the CXCR4-activated MEK/ERK and PI3K/AKT pathways

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2018.9444

    Isolation and identification of OPCs. (A) Immunostaining demonstrated that OPCs were positive for NG2. (B) Immunostaining demonstrated that OPCs were positive for PDGFR-α. (C) Immunostaining showed that the differentiated OPCs were positive for O4. (D) Immunostaining showed that the differentiated OPCs were positive for MBP. Nuclei were stained with DAPI (blue). NG2, neural/glial antigen 2; PDGFR-α, platelet-derived growth factor receptor-α; OPCs, oligodendrocyte precursor cells; O4, oligodendrocyte marker O4; MBP, myelin basic protein.
    Figure Legend Snippet: Isolation and identification of OPCs. (A) Immunostaining demonstrated that OPCs were positive for NG2. (B) Immunostaining demonstrated that OPCs were positive for PDGFR-α. (C) Immunostaining showed that the differentiated OPCs were positive for O4. (D) Immunostaining showed that the differentiated OPCs were positive for MBP. Nuclei were stained with DAPI (blue). NG2, neural/glial antigen 2; PDGFR-α, platelet-derived growth factor receptor-α; OPCs, oligodendrocyte precursor cells; O4, oligodendrocyte marker O4; MBP, myelin basic protein.

    Techniques Used: Isolation, Immunostaining, Staining, Derivative Assay, Marker

    9) Product Images from "4-AMINOPYRIDINE ATTENUATES MUSCLE ATROPHY AFTER SCIATIC NERVE CRUSH INJURY IN MICE"

    Article Title: 4-AMINOPYRIDINE ATTENUATES MUSCLE ATROPHY AFTER SCIATIC NERVE CRUSH INJURY IN MICE

    Journal: Muscle & nerve

    doi: 10.1002/mus.26516

    In vivo  4-AP treatment increases Pax7- and Ki67-expressing SCs in TA and EDL muscles of injured limb.  (A)  Representative Pax7/Ki67/DAPI IF images in CTL muscles at day 0; each image represents 9 images from 3 different mice.  (B)  Representative images of Pax7 (red), Ki67 (green), and DAPI (blue) in 4-AP-treated group at day 14 postinjury; each image represents 9 images from 3 different mice.  (C)  Representative Pax7/Ki67/DAPI IF images in Sal and 4-AP-treated groups at days 7 and 14 postinjury. Frozen muscle cross-sections were incubated simultaneously with 2 different primary antibodies (Pax7 mouse antibody and Ki67 rabbit antibody) and 2 different secondary antibodies (AlexaFluor 594-conjugated goat anti-mouse IgG and AlexaFluor 488-conjugated goat anti-rabbit IgM). In addition to individual red (Pax7) or green (Ki67) color, combination of red and green colors resulted in yellow cells; each image represents 9 images from 3 different mice.  (D,E)  The pooled number of Pax7 +  ( D ) and Ki67 +  ( E ) cells in uninjured and injured groups with or without treatment;  n  = 3/group. * P
    Figure Legend Snippet: In vivo 4-AP treatment increases Pax7- and Ki67-expressing SCs in TA and EDL muscles of injured limb. (A) Representative Pax7/Ki67/DAPI IF images in CTL muscles at day 0; each image represents 9 images from 3 different mice. (B) Representative images of Pax7 (red), Ki67 (green), and DAPI (blue) in 4-AP-treated group at day 14 postinjury; each image represents 9 images from 3 different mice. (C) Representative Pax7/Ki67/DAPI IF images in Sal and 4-AP-treated groups at days 7 and 14 postinjury. Frozen muscle cross-sections were incubated simultaneously with 2 different primary antibodies (Pax7 mouse antibody and Ki67 rabbit antibody) and 2 different secondary antibodies (AlexaFluor 594-conjugated goat anti-mouse IgG and AlexaFluor 488-conjugated goat anti-rabbit IgM). In addition to individual red (Pax7) or green (Ki67) color, combination of red and green colors resulted in yellow cells; each image represents 9 images from 3 different mice. (D,E) The pooled number of Pax7 + ( D ) and Ki67 + ( E ) cells in uninjured and injured groups with or without treatment; n = 3/group. * P

    Techniques Used: In Vivo, Expressing, Mouse Assay, Incubation

    10) Product Images from "Benefits and Pitfalls of Secondary Antibodies: Why Choosing the Right Secondary Is of Primary Importance"

    Article Title: Benefits and Pitfalls of Secondary Antibodies: Why Choosing the Right Secondary Is of Primary Importance

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0038313

    HL 2°Abs show a bias for immunohistochemistry labeling with mAbs of different IgG subclasses. Rat brain sections were labeled with the same concentrations of a single mAb, and a rabbit anti-Kv2.1 pAb, followed by detection with SCS (left column) or HL (right column) 2°Abs, (red), and anti-rabbit IgG (green), each at 1 µg/ml. Top row: anti-Kv4.2 IgG1; middle row: anti-BK channel IgG2a; and bottom row: anti-Kv1.2 IgG2b. Each row was imaged at the same exposure times. Scale bar = 50 µm for panels in top two rows, and 25 µm for panels in bottom row.
    Figure Legend Snippet: HL 2°Abs show a bias for immunohistochemistry labeling with mAbs of different IgG subclasses. Rat brain sections were labeled with the same concentrations of a single mAb, and a rabbit anti-Kv2.1 pAb, followed by detection with SCS (left column) or HL (right column) 2°Abs, (red), and anti-rabbit IgG (green), each at 1 µg/ml. Top row: anti-Kv4.2 IgG1; middle row: anti-BK channel IgG2a; and bottom row: anti-Kv1.2 IgG2b. Each row was imaged at the same exposure times. Scale bar = 50 µm for panels in top two rows, and 25 µm for panels in bottom row.

    Techniques Used: Immunohistochemistry, Labeling

    HL 2°Abs exhibit background and detection bias independent of 1° and 2°Ab concentrations. (A) FLISAs showing detection of different concentrations of IgG1 (K14/39, squares), IgG2a (L76/36, circles), and IgG2b (K14/16, triangles) mAbs as indicated by the values on the X-axes, with HL 2°Ab (top row), and respective SCS 2°Abs (middle row), at the concentrations indicated above the columns. Bottom row shows data from the graphs in the top row normalized to values for the IgG1 mAb. (B) HL bias is seen at all 2°Ab concentrations tested in transiently transfected COS-1 cells. Immunofluorescence labeling of Kv1.2-expressing COS-1 cells, probed with 5 µg/mL of IgG1 (K14/39, squares), IgG2a (L76/36, circles), and IgG2b (K14/16, triangles) mAbs and different amounts of HL 2°Ab (red), and the respective SCS 2°Abs (green), as indicated on the X-axis. The Y-axis is the red∶green (HL∶SCS) fluorescence ratio (in arbitrary units). (C) Immunoblots showing lack of crossreactivity in SCS 2°Ab detection of antigens loaded at great excess. Recombinant GST fusion proteins containing different amounts of Kv1.2 and PSD95 antigens, and GST alone, were size fractionated on a single SDS gel and transferred to an immunoblot. Amounts loaded of GST-PSD95 ranged from 4–972 ng, as indicated below lower left panel, and for GST-Kv1.2 and GST alone from 972–4 ng, as indicated below lower right panel. The immunoblot was simultaneously probed with anti-Kv1.2 K14/16 (IgG2b, red), anti-PSD95 K28/43 (IgG2a, blue) and anti-GST N100/13 (IgG1, green), and corresponding SCS 2°Abs. Lane to left of top left panel shows molecular weight standards in kDa. Image reveals a lack of crossreactivity between SCS 2°Abs and bound 1°Abs even under conditions of excess antigen.
    Figure Legend Snippet: HL 2°Abs exhibit background and detection bias independent of 1° and 2°Ab concentrations. (A) FLISAs showing detection of different concentrations of IgG1 (K14/39, squares), IgG2a (L76/36, circles), and IgG2b (K14/16, triangles) mAbs as indicated by the values on the X-axes, with HL 2°Ab (top row), and respective SCS 2°Abs (middle row), at the concentrations indicated above the columns. Bottom row shows data from the graphs in the top row normalized to values for the IgG1 mAb. (B) HL bias is seen at all 2°Ab concentrations tested in transiently transfected COS-1 cells. Immunofluorescence labeling of Kv1.2-expressing COS-1 cells, probed with 5 µg/mL of IgG1 (K14/39, squares), IgG2a (L76/36, circles), and IgG2b (K14/16, triangles) mAbs and different amounts of HL 2°Ab (red), and the respective SCS 2°Abs (green), as indicated on the X-axis. The Y-axis is the red∶green (HL∶SCS) fluorescence ratio (in arbitrary units). (C) Immunoblots showing lack of crossreactivity in SCS 2°Ab detection of antigens loaded at great excess. Recombinant GST fusion proteins containing different amounts of Kv1.2 and PSD95 antigens, and GST alone, were size fractionated on a single SDS gel and transferred to an immunoblot. Amounts loaded of GST-PSD95 ranged from 4–972 ng, as indicated below lower left panel, and for GST-Kv1.2 and GST alone from 972–4 ng, as indicated below lower right panel. The immunoblot was simultaneously probed with anti-Kv1.2 K14/16 (IgG2b, red), anti-PSD95 K28/43 (IgG2a, blue) and anti-GST N100/13 (IgG1, green), and corresponding SCS 2°Abs. Lane to left of top left panel shows molecular weight standards in kDa. Image reveals a lack of crossreactivity between SCS 2°Abs and bound 1°Abs even under conditions of excess antigen.

    Techniques Used: Transfection, Immunofluorescence, Labeling, Expressing, Fluorescence, Western Blot, Recombinant, SDS-Gel, Molecular Weight

    HL 2°Abs show a bias for mAbs of different IgG subclasses in a variety of applications. (A) A single immunoblot containing samples of crude rat brain membranes (RBM, 50 µg protein) and extracts of transfected COS-1 cells expressing individual target proteins, or from control cells transfected with an empty plasmid as labeled, probed with anti-PSD-95 (IgG2a), anti-Kv1.2 (IgG2b) and anti-Kv2.1 (IgG1) mAbs, and HL 2°Ab (green), and a cocktail (1∶1∶1) of SCS anti-IgG1, -IgG2a and -IgG2b 2°Abs (red). Multicolor panel is original immunoblot; single color panels are images of separated colors. Changes in tint reflect bias of HL for (more green) IgG2a > IgG2b > IgG1 (more red). Lane to left shows molecular weight standards in kDa. (B) FLISAs show that IgG subclass bias of HL is present at all concentrations of 1°Abs. Left panel: SCS 2°Abs (each at 1 µg/ml). Right panel: HL 2°Ab. Circles: L76/36 IgG2a; triangles; K14/16 IgG2b; squares: K14/39 IgG1. (C) IgG subclass bias is also present in immunofluorescence labeling of Kv1.2-expressing COS-1 cells. Cells were labeled with mAb as noted, and HL 2°Ab (red), and SCS 2°Abs (green) as detailed in Methods . Changes in red∶green tint reflect bias of HL for (more red) IgG2a > IgG2b > IgG1 (more green). Scale bar = 100 µm. Panel to right is quantitation of immunocytochemistry results from three fields each of three independent samples.
    Figure Legend Snippet: HL 2°Abs show a bias for mAbs of different IgG subclasses in a variety of applications. (A) A single immunoblot containing samples of crude rat brain membranes (RBM, 50 µg protein) and extracts of transfected COS-1 cells expressing individual target proteins, or from control cells transfected with an empty plasmid as labeled, probed with anti-PSD-95 (IgG2a), anti-Kv1.2 (IgG2b) and anti-Kv2.1 (IgG1) mAbs, and HL 2°Ab (green), and a cocktail (1∶1∶1) of SCS anti-IgG1, -IgG2a and -IgG2b 2°Abs (red). Multicolor panel is original immunoblot; single color panels are images of separated colors. Changes in tint reflect bias of HL for (more green) IgG2a > IgG2b > IgG1 (more red). Lane to left shows molecular weight standards in kDa. (B) FLISAs show that IgG subclass bias of HL is present at all concentrations of 1°Abs. Left panel: SCS 2°Abs (each at 1 µg/ml). Right panel: HL 2°Ab. Circles: L76/36 IgG2a; triangles; K14/16 IgG2b; squares: K14/39 IgG1. (C) IgG subclass bias is also present in immunofluorescence labeling of Kv1.2-expressing COS-1 cells. Cells were labeled with mAb as noted, and HL 2°Ab (red), and SCS 2°Abs (green) as detailed in Methods . Changes in red∶green tint reflect bias of HL for (more red) IgG2a > IgG2b > IgG1 (more green). Scale bar = 100 µm. Panel to right is quantitation of immunocytochemistry results from three fields each of three independent samples.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Labeling, Molecular Weight, Immunofluorescence, Quantitation Assay, Immunocytochemistry

    HL detection bias is seen in 2°Ab preparations from different suppliers, with different fluorophores, and with enzyme conjugates. (A) Kv1.2-transfected COS-1 cells were labeled with 1°Ab as in Figure 4C , and HL 2°Ab and the respective SCS 2°Abs, and the ratios of fluorescence intensities from three fields each of three independent samples normalized to the HL/IgG1 ratio. Letters are supplier (L = Life Technologies, R = Rockland), numbers are Alex or DyLight fluorophore conjugates; high: highly adsorbed; fab: F(ab′) 2 fragment of HL ( e.g. , L488 SCS is Life Technologies Alexa 488 conjugated SCS). 4/09 and 7/11 refer to two lots of Life Technologies HL. (B) FLISAs showing detection bias of 2°Abs is present at all 2°Ab concentrations. Upper left: Life Technologies HL. Upper right: Life Technologies SCS. Lower left: Jackson ImmunoResearch HL. Lower right: Jackson ImmunoResearch HL (highly cross-adsorbed). (C) HRP conjugated HL secondaries show detection bias by immunoblot. Purified mAb IgG preparations were analyzed by reducing SDS-PAGE and coomassie blue staining (CB), or immunoblotting and detection with two different HRP-conjugated H+L 2°Abs and ECL. HL: Kirkegaard Perry Laboratories. HL*: Antibodies Incorporated. Note subclass-specific differences in detection of heavy chain (HC) but not light chain (LC) bands in IgG preparations.
    Figure Legend Snippet: HL detection bias is seen in 2°Ab preparations from different suppliers, with different fluorophores, and with enzyme conjugates. (A) Kv1.2-transfected COS-1 cells were labeled with 1°Ab as in Figure 4C , and HL 2°Ab and the respective SCS 2°Abs, and the ratios of fluorescence intensities from three fields each of three independent samples normalized to the HL/IgG1 ratio. Letters are supplier (L = Life Technologies, R = Rockland), numbers are Alex or DyLight fluorophore conjugates; high: highly adsorbed; fab: F(ab′) 2 fragment of HL ( e.g. , L488 SCS is Life Technologies Alexa 488 conjugated SCS). 4/09 and 7/11 refer to two lots of Life Technologies HL. (B) FLISAs showing detection bias of 2°Abs is present at all 2°Ab concentrations. Upper left: Life Technologies HL. Upper right: Life Technologies SCS. Lower left: Jackson ImmunoResearch HL. Lower right: Jackson ImmunoResearch HL (highly cross-adsorbed). (C) HRP conjugated HL secondaries show detection bias by immunoblot. Purified mAb IgG preparations were analyzed by reducing SDS-PAGE and coomassie blue staining (CB), or immunoblotting and detection with two different HRP-conjugated H+L 2°Abs and ECL. HL: Kirkegaard Perry Laboratories. HL*: Antibodies Incorporated. Note subclass-specific differences in detection of heavy chain (HC) but not light chain (LC) bands in IgG preparations.

    Techniques Used: Transfection, Labeling, Fluorescence, Purification, SDS Page, Staining

    Analysis of knockout mouse tissue reveals increased background of HL 2°Abs. Sections of brains from WT and Kv2.1 knockout (KO) mice were labeled with an anti-Kv2.1 IgG1 mAb, or in vehicle alone (bottom row, no 1°Ab), followed by simultaneous detection with both HL (green) and IgG1-specific (red) 2°Abs. Columns represent samples with different [2°Ab] as in column header. All samples were imaged using identical exposure times. Note that the panels in the top row are the same as those in the WT row but showing the green channel only. Scale bar = 25 µm.
    Figure Legend Snippet: Analysis of knockout mouse tissue reveals increased background of HL 2°Abs. Sections of brains from WT and Kv2.1 knockout (KO) mice were labeled with an anti-Kv2.1 IgG1 mAb, or in vehicle alone (bottom row, no 1°Ab), followed by simultaneous detection with both HL (green) and IgG1-specific (red) 2°Abs. Columns represent samples with different [2°Ab] as in column header. All samples were imaged using identical exposure times. Note that the panels in the top row are the same as those in the WT row but showing the green channel only. Scale bar = 25 µm.

    Techniques Used: Knock-Out, Mouse Assay, Labeling

    SCS 2°Abs yield robust and reliable simultaneous triple labeling with three mAbs on immunoblots and in rat brain sections. (A) A single immunoblot containing samples of crude rat brain membranes (RBM, 50 µg protein) and extracts of transfected COS-1 cells expressing individual target proteins, or from control cells transfected with an empty plasmid, probed with anti-PSD-95 (IgG2a, blue), anti-Kv1.2 (IgG2b, red) and anti-Kv2.1 (IgG1, green), and SCS 2°Abs. Multicolor panel is original immunoblot; single color panels are images of separated colors. Lane to left shows molecular weight standards in kDa. Note differential post-translational modification of target proteins in brain versus heterologous cells alters their relative electrophoretic mobility. B–E. Images show specific and non-overlapping labeling for (B) Kv4.2 (green), (C) QKI (red), (D) and BK channels (blue), and (E) merge of all three, in a rat brain section, showing the region containing the entire cerebellum. Inset in E shows boxed area of cerebellar cortex. Labels mark the molecular layer (ML), Purkinje cell layer (PCL), and granule cell layer (GCL). Scale bar on Panel E = 500 µm.
    Figure Legend Snippet: SCS 2°Abs yield robust and reliable simultaneous triple labeling with three mAbs on immunoblots and in rat brain sections. (A) A single immunoblot containing samples of crude rat brain membranes (RBM, 50 µg protein) and extracts of transfected COS-1 cells expressing individual target proteins, or from control cells transfected with an empty plasmid, probed with anti-PSD-95 (IgG2a, blue), anti-Kv1.2 (IgG2b, red) and anti-Kv2.1 (IgG1, green), and SCS 2°Abs. Multicolor panel is original immunoblot; single color panels are images of separated colors. Lane to left shows molecular weight standards in kDa. Note differential post-translational modification of target proteins in brain versus heterologous cells alters their relative electrophoretic mobility. B–E. Images show specific and non-overlapping labeling for (B) Kv4.2 (green), (C) QKI (red), (D) and BK channels (blue), and (E) merge of all three, in a rat brain section, showing the region containing the entire cerebellum. Inset in E shows boxed area of cerebellar cortex. Labels mark the molecular layer (ML), Purkinje cell layer (PCL), and granule cell layer (GCL). Scale bar on Panel E = 500 µm.

    Techniques Used: Labeling, Western Blot, Transfection, Expressing, Plasmid Preparation, Molecular Weight, Modification

    11) Product Images from "The Ox40/Ox40 Ligand Pathway Promotes Pathogenic Th Cell Responses, Plasmablast Accumulation, and Lupus Nephritis in NZB/W F1 Mice"

    Article Title: The Ox40/Ox40 Ligand Pathway Promotes Pathogenic Th Cell Responses, Plasmablast Accumulation, and Lupus Nephritis in NZB/W F1 Mice

    Journal: The Journal of Immunology Author Choice

    doi: 10.4049/jimmunol.1700608

    Anti-Ox40 agonist mAb treatment exacerbates renal disease and IgM deposition in NZB/W F1 mice. Renal disease monitoring following anti-Ox40 agonist mAb (red) and a control Ig (blue) treatment (10 mg/kg, three times per week for 3 wk) in NZB/W F1 mice. Black arrows (Rx) refer to treatment days. The age and proteinuria status of each cohort (at the time of first treatment) is indicated at the top of each plot. ( A ) Thirteen-week-old proteinuria-free NZB/W F1 mice at treatment start. ( B ) Three independent cohorts (21-, 26-, and 27-wk-old) of NZB/W F1 mice with proteinuria 30–100 mg/dl at treatment start. ( C ) Representative H E- and PAS-stained kidney sections (left and middle panels) highlighting glomeruli (top panels), periarterial regions (middle panels), and tubulointerstitium (bottom panels). Scale bars, 100 μm. Corresponding disease severity scores (right panels). Group means are plotted in black ( n = 36 per group combined). ( D ) Representative images from frozen kidney sections (left panels) stained for glomerular deposits of IgM (top panel), IgG (middle panel), and C3 (bottom panel). Scale bars, 50 μm. Corresponding signal intensity in the cortex regions is presented as pixel intensity/μM 2 of cortex with group mean ± SD from two independent cohorts ( n = 25–28 per group, combined) (right panels). * p
    Figure Legend Snippet: Anti-Ox40 agonist mAb treatment exacerbates renal disease and IgM deposition in NZB/W F1 mice. Renal disease monitoring following anti-Ox40 agonist mAb (red) and a control Ig (blue) treatment (10 mg/kg, three times per week for 3 wk) in NZB/W F1 mice. Black arrows (Rx) refer to treatment days. The age and proteinuria status of each cohort (at the time of first treatment) is indicated at the top of each plot. ( A ) Thirteen-week-old proteinuria-free NZB/W F1 mice at treatment start. ( B ) Three independent cohorts (21-, 26-, and 27-wk-old) of NZB/W F1 mice with proteinuria 30–100 mg/dl at treatment start. ( C ) Representative H E- and PAS-stained kidney sections (left and middle panels) highlighting glomeruli (top panels), periarterial regions (middle panels), and tubulointerstitium (bottom panels). Scale bars, 100 μm. Corresponding disease severity scores (right panels). Group means are plotted in black ( n = 36 per group combined). ( D ) Representative images from frozen kidney sections (left panels) stained for glomerular deposits of IgM (top panel), IgG (middle panel), and C3 (bottom panel). Scale bars, 50 μm. Corresponding signal intensity in the cortex regions is presented as pixel intensity/μM 2 of cortex with group mean ± SD from two independent cohorts ( n = 25–28 per group, combined) (right panels). * p

    Techniques Used: Mouse Assay, Staining

    Ox40-stimulated splenic T cell response, plasmablast accumulation, and Ig effects. Quantification of total serum IgM concentration ( A ) and total serum IgG1, IgG2a, IgG2b, and IgG3 concentrations ( B ) following anti-Ox40 mAb treatment presented as mean ± SD combined from three independent cohorts ( n = 24 for IgG1 and 36 for all other groups, combined). ( C – E ) Representative flow cytometry plots (left panels) and summary data (right panels) from NZB/W F1 spleen preparations presented with mean ± SD. (C) Frequency of activation/memory cell markers CD44 and CD62L on Tconv cells (gated live CD45 + CD3 + CD4 + Foxp3 − ). (D) Frequency and total number of CXCR5 + ICOS + Tfh cells (gated CD3 + CD4 + ). (E) Frequency and total number of plasmablast cells (CD138 + ). ( F ) Total number of IgM + CD138 + plasmablast cells. * p
    Figure Legend Snippet: Ox40-stimulated splenic T cell response, plasmablast accumulation, and Ig effects. Quantification of total serum IgM concentration ( A ) and total serum IgG1, IgG2a, IgG2b, and IgG3 concentrations ( B ) following anti-Ox40 mAb treatment presented as mean ± SD combined from three independent cohorts ( n = 24 for IgG1 and 36 for all other groups, combined). ( C – E ) Representative flow cytometry plots (left panels) and summary data (right panels) from NZB/W F1 spleen preparations presented with mean ± SD. (C) Frequency of activation/memory cell markers CD44 and CD62L on Tconv cells (gated live CD45 + CD3 + CD4 + Foxp3 − ). (D) Frequency and total number of CXCR5 + ICOS + Tfh cells (gated CD3 + CD4 + ). (E) Frequency and total number of plasmablast cells (CD138 + ). ( F ) Total number of IgM + CD138 + plasmablast cells. * p

    Techniques Used: Concentration Assay, Flow Cytometry, Cytometry, Activation Assay

    12) Product Images from "Critical role of activation induced cytidine deaminase in Experimental Autoimmune Encephalomyelitis"

    Article Title: Critical role of activation induced cytidine deaminase in Experimental Autoimmune Encephalomyelitis

    Journal: Autoimmunity

    doi: 10.3109/08916934.2012.750301

    Indirect fluorescence for binding of serum antibody to normal brain tissue. Serum from EAE affected WT mice show diffuse, strong white matter associated signal consistent with the presence of MOG specific IgG1. By contrast, corresponding signal is absent using serum from MOG induced KO mice, or healthy controls. There is no difference of signal intensity or distribution for IgM binding to brain, regardless of MOG induction or genotype. Representative images are shown.
    Figure Legend Snippet: Indirect fluorescence for binding of serum antibody to normal brain tissue. Serum from EAE affected WT mice show diffuse, strong white matter associated signal consistent with the presence of MOG specific IgG1. By contrast, corresponding signal is absent using serum from MOG induced KO mice, or healthy controls. There is no difference of signal intensity or distribution for IgM binding to brain, regardless of MOG induction or genotype. Representative images are shown.

    Techniques Used: Fluorescence, Binding Assay, Mouse Assay

    Generation and functional characterization of Aicda deficient mice. A) Gene targeting strategy used to delete the catalytic domain (exon-3) of Aicda locus. The structure of the AID domains is shown in top. The location of the homology arms in the targeting vector is indicated. The schematic of targeting construct used to delete the catalytic domain of AID (exon-3) is shown in the middle. The solid triangles represent Cre recombination sites and the Neomycin cassette was subsequently removed by expression of Cre-recombinase. Representative WT or KO PCR products using 3′ or 5′ arm analysis at the Aicda locus is shown in the right panel. The size of Aicda KO PCR product is indicated and corresponds to the modified locus. Notice that one primer is located in the Neomycin cassette and therefore only detects the targeted Aicda locus. B) FACS analysis of LPS/IL4 stimulated WT or AID KO B cells in vitro. Percentage of IgM or IgG1 are indicated in each quadrant. B220-FITC is used as a B-cell marker.
    Figure Legend Snippet: Generation and functional characterization of Aicda deficient mice. A) Gene targeting strategy used to delete the catalytic domain (exon-3) of Aicda locus. The structure of the AID domains is shown in top. The location of the homology arms in the targeting vector is indicated. The schematic of targeting construct used to delete the catalytic domain of AID (exon-3) is shown in the middle. The solid triangles represent Cre recombination sites and the Neomycin cassette was subsequently removed by expression of Cre-recombinase. Representative WT or KO PCR products using 3′ or 5′ arm analysis at the Aicda locus is shown in the right panel. The size of Aicda KO PCR product is indicated and corresponds to the modified locus. Notice that one primer is located in the Neomycin cassette and therefore only detects the targeted Aicda locus. B) FACS analysis of LPS/IL4 stimulated WT or AID KO B cells in vitro. Percentage of IgM or IgG1 are indicated in each quadrant. B220-FITC is used as a B-cell marker.

    Techniques Used: Functional Assay, Mouse Assay, Plasmid Preparation, Construct, Expressing, Polymerase Chain Reaction, Modification, FACS, In Vitro, Marker

    Characterization of anti-MOG antibodies using ELISA at day zero and 28. A) IgG anti-rhMOG, B) IgM anti-rhMOG, C) IgG anti-MOG peptide 35–55, D) IgM anti-MOG peptide 35–55. Mixed day 28 sera of WT or KO immunized mice were used as standard (arbitrarily defined as 100 U) for IgG or IgM antibodies, respectively. E) IgM binding to rhMOG coated at different concentrations. Sera from 8 mice were pooled for each of the following sample sets: Day 0 WT, Day 0 KO, Day 28 WT, and Day 28 KO. Pooled mice sera IgM Abs normalized at 0.1 mg/ml in the assay. Anti-mouse IgM was used as detection to exclusively measure the binding of IgM antibodies in serum.
    Figure Legend Snippet: Characterization of anti-MOG antibodies using ELISA at day zero and 28. A) IgG anti-rhMOG, B) IgM anti-rhMOG, C) IgG anti-MOG peptide 35–55, D) IgM anti-MOG peptide 35–55. Mixed day 28 sera of WT or KO immunized mice were used as standard (arbitrarily defined as 100 U) for IgG or IgM antibodies, respectively. E) IgM binding to rhMOG coated at different concentrations. Sera from 8 mice were pooled for each of the following sample sets: Day 0 WT, Day 0 KO, Day 28 WT, and Day 28 KO. Pooled mice sera IgM Abs normalized at 0.1 mg/ml in the assay. Anti-mouse IgM was used as detection to exclusively measure the binding of IgM antibodies in serum.

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

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    Article Title: Heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs) function as endocytic receptors for an internalizing anti-nucleic acid antibody
    Article Snippet: .. After washing, cells were incubated with a secondary antibody mixture comprising TRITC-conjugated goat anti-rabbit IgG (Sigma, cat# T6778) and an Alexa Fluor 488-conjugated goat anti-mouse IgM/μ chain-specific antibody (Invitrogen, cat# A21042) diluted in surface buffer for 1–2 h at RT. .. Each incubation step was followed by three washes with cold PBS.

    other:

    Article Title: 4-AMINOPYRIDINE ATTENUATES MUSCLE ATROPHY AFTER SCIATIC NERVE CRUSH INJURY IN MICE
    Article Snippet: Additional Materials UsedAntibodies used were anti-Pax7 (mouse IgG1, dilution 1:100, catalog No. AB_528428; Developmental Studies Hybridoma Bank, Iowa City, Iowa), antibody of laminin (rat or rabbit, dilution 1:1,500, catalog No. L0663 or L939; Sigma-Aldrich), anti-Ki67 (dilution 1:400, catalog No. 9129S; Cell Signaling Technology, Danvers, Massachusetts), anti-myogenin (F5D, dilution 1:100, sc-12732; Santa Cruz Bio-technology, Dallas, Texas), anti-GAPDH (dilution 1:2,500, ab9485; Abcam, Cambridge, Massachusetts), AffiniPure Fab fragment goat anti-mouse IgG (H + L, AFFGAI, 0.1 mg/ml; catalog No. 115–007-003; Jackson ImmunoResearch), AlexaFluor 594-conjugated goat anti-mouse IgG (H + L, dilution 1:1,500, catalog No. A-11032,; Life Technologies, Grand Island, New York), AlexaFluor 488-conjugated goat anti-mouse IgM (dilution 1:1,500, catalog No. A-21042; Life Technologies), and AlexaFluor 647-conjugated goat anti-rabbit or anti-mouse antibodies (dilution 1:1,500, catalog No. A-21244 or A-21235; Life Technologies).

    Western Blot:

    Article Title: Independent modes of disease repair by AIM protein distinguished in AIM-felinized mice
    Article Snippet: .. Primary antibodies: AIM (rab2 rabbit polyclonal for mice AIM in western blotting and immunohistochemistry, PAC-11 rabbit polyclonal for feline AIM in western blotting, and clone #33 mouse monoclonal for feline AIM in western blotting and immunohistochemistry; established in our laboratory), IgM (A21044, goat polyclonal, Alexa 594 conjugated, Thermo Fisher Scientific, Waltham, MA), F4/80 (clone: BM8 for immunohistochemistry; Thermo Fisher Scientific), CD36 (clone: MF3 for immunohistochemistry; Abcam, Cambridge, UK) and gp73 (goat polyclonal for immunohistochemistry; Santa Cruz, Dallas, TX). .. Secondary Antibodies: goat anti-rabbit IgG (H + L) horseradish peroxidase (HRP) conjugated (Thermo Fisher Scientific), Alexa Fluor 488- or 594-conjugated anti-rat IgG, and Alexa Fluor 647-conjugated anti-rabbit IgG (Thermo Fisher Scientific).

    Staining:

    Article Title: The Ox40/Ox40 Ligand Pathway Promotes Pathogenic Th Cell Responses, Plasmablast Accumulation, and Lupus Nephritis in NZB/W F1 Mice
    Article Snippet: .. Glomerular IC deposits were visualized on 5-μm acetone-fixed OCT-embedded kidney sections by direct immunofluorescence staining using Alexa Fluor 488–conjugated donkey anti-mouse IgG (cat. no. A-21202; Invitrogen), goat anti-mouse IgM (cat. no. A-21042; Invitrogen), or fluorescein-conjugated goat anti-complement C3 (cat. no. 55510; MP Biomedicals). .. Naive donkey or goat IgG (Jackson ImmunoResearch) was used in parallel as nonspecific isotype controls and were negative.

    Immunofluorescence:

    Article Title: The Ox40/Ox40 Ligand Pathway Promotes Pathogenic Th Cell Responses, Plasmablast Accumulation, and Lupus Nephritis in NZB/W F1 Mice
    Article Snippet: .. Glomerular IC deposits were visualized on 5-μm acetone-fixed OCT-embedded kidney sections by direct immunofluorescence staining using Alexa Fluor 488–conjugated donkey anti-mouse IgG (cat. no. A-21202; Invitrogen), goat anti-mouse IgM (cat. no. A-21042; Invitrogen), or fluorescein-conjugated goat anti-complement C3 (cat. no. 55510; MP Biomedicals). .. Naive donkey or goat IgG (Jackson ImmunoResearch) was used in parallel as nonspecific isotype controls and were negative.

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    Thermo Fisher goat anti mouse igm heavy chain cross adsorbed secondary antibody
    States of inflammation and fibrosis in the liver in the absence of serum <t>IgM-free</t> <t>AIM.</t> ( a ) The mRNA levels of various genes responsive to different types of stresses (i.e., endoplasmic reticulum, mitochondrial, or oxidative stress) addressed by qPCR using RNA from the whole liver of AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 5–6 for 12-week HFD per group). Error bar indicates the SEM. (b,c) The mRNA levels of inflammatory cytokine genes in the liver, as assessed by qPCR using the RNA as in ( a ). (d ) Representative photomicrographs of liver from AIM-felinized, WT, AIM −/− , and Δsμ mice fed an HFD for 12 weeks stained with Sirius red Stain. Scale bars, 100 μm. (e) The mRNA levels of fibrogenetic genes in the liver, as assessed by qPCR using the same RNA as in ( a ). In (a – c) and (e) , *is used to represent the statistical significance between the values of each mouse strain group within the same period, whereas # is attached to the bar of 12 w when the value of 12 w was significantly changed compared with that of 0 w in the same mouse strain group.
    Goat Anti Mouse Igm Heavy Chain Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 717 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    States of inflammation and fibrosis in the liver in the absence of serum IgM-free AIM. ( a ) The mRNA levels of various genes responsive to different types of stresses (i.e., endoplasmic reticulum, mitochondrial, or oxidative stress) addressed by qPCR using RNA from the whole liver of AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 5–6 for 12-week HFD per group). Error bar indicates the SEM. (b,c) The mRNA levels of inflammatory cytokine genes in the liver, as assessed by qPCR using the RNA as in ( a ). (d ) Representative photomicrographs of liver from AIM-felinized, WT, AIM −/− , and Δsμ mice fed an HFD for 12 weeks stained with Sirius red Stain. Scale bars, 100 μm. (e) The mRNA levels of fibrogenetic genes in the liver, as assessed by qPCR using the same RNA as in ( a ). In (a – c) and (e) , *is used to represent the statistical significance between the values of each mouse strain group within the same period, whereas # is attached to the bar of 12 w when the value of 12 w was significantly changed compared with that of 0 w in the same mouse strain group.

    Journal: Scientific Reports

    Article Title: Independent modes of disease repair by AIM protein distinguished in AIM-felinized mice

    doi: 10.1038/s41598-018-31580-6

    Figure Lengend Snippet: States of inflammation and fibrosis in the liver in the absence of serum IgM-free AIM. ( a ) The mRNA levels of various genes responsive to different types of stresses (i.e., endoplasmic reticulum, mitochondrial, or oxidative stress) addressed by qPCR using RNA from the whole liver of AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 5–6 for 12-week HFD per group). Error bar indicates the SEM. (b,c) The mRNA levels of inflammatory cytokine genes in the liver, as assessed by qPCR using the RNA as in ( a ). (d ) Representative photomicrographs of liver from AIM-felinized, WT, AIM −/− , and Δsμ mice fed an HFD for 12 weeks stained with Sirius red Stain. Scale bars, 100 μm. (e) The mRNA levels of fibrogenetic genes in the liver, as assessed by qPCR using the same RNA as in ( a ). In (a – c) and (e) , *is used to represent the statistical significance between the values of each mouse strain group within the same period, whereas # is attached to the bar of 12 w when the value of 12 w was significantly changed compared with that of 0 w in the same mouse strain group.

    Article Snippet: Primary antibodies: AIM (rab2 rabbit polyclonal for mice AIM in western blotting and immunohistochemistry, PAC-11 rabbit polyclonal for feline AIM in western blotting, and clone #33 mouse monoclonal for feline AIM in western blotting and immunohistochemistry; established in our laboratory), IgM (A21044, goat polyclonal, Alexa 594 conjugated, Thermo Fisher Scientific, Waltham, MA), F4/80 (clone: BM8 for immunohistochemistry; Thermo Fisher Scientific), CD36 (clone: MF3 for immunohistochemistry; Abcam, Cambridge, UK) and gp73 (goat polyclonal for immunohistochemistry; Santa Cruz, Dallas, TX).

    Techniques: Real-time Polymerase Chain Reaction, Mouse Assay, Staining

    Effect of serum IgM-free AIM on obesity and liver steatosis. ( a,b) Weights from AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 6–9 for 12-week HFD per group). Error bar indicates the SEM. (a) Body weights. (b ) Weights of epidydimal adipose tissues. (c) Representative photomicrographs of epididymal fat tissues from AIM-felinized, WT, AIM −/− , and Δsµ mice fed an HFD for 12 weeks stained with H E. Adipocyte sizes of 50 independent adipocytes in different areas were evaluated. Results are presented as average ± SEM (in μm 2 ). Scale bars, 100 μm. ( d ) Representative photomicrographs of epididymal fat tissues from AIM-felinized, WT, and AIM −/− mice (fed an HFD for 12 weeks) stained for AIM (blue), F4/80 (macrophage marker; green), and IgM (red, WT and AIM −/− mice. See Supplementary Fig. 1 for AIM-felinized mice). Yellow arrows represent where IgM-free AIM signals exist, while red arrows indicate where AIM is co-stained with IgM. Scale bars, 100 μm. (e) The mRNA levels of AIM and F4/80 were assessed by qPCR using RNA isolated from epididymal fat in WT mice before or after being fed an HFD for 12 weeks. Values were normalized to those of GAPDH and presented as the expression relative to that of AIM from lean WT mice liver and of F4/80 from fat tissues before being fed an HFD (n = 4 per group). Error bar indicates the SEM. (f) Representative photomicrographs of liver from AIM-felinized, WT, AIM −/− , and Δsµ mice fed an HFD for 12 weeks stained with H E. Scale bars, 100 µm. (g) Liver weights and TG contents from AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 6–9 for 12-week HFD per group). Error bar indicates the SEM.

    Journal: Scientific Reports

    Article Title: Independent modes of disease repair by AIM protein distinguished in AIM-felinized mice

    doi: 10.1038/s41598-018-31580-6

    Figure Lengend Snippet: Effect of serum IgM-free AIM on obesity and liver steatosis. ( a,b) Weights from AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 6–9 for 12-week HFD per group). Error bar indicates the SEM. (a) Body weights. (b ) Weights of epidydimal adipose tissues. (c) Representative photomicrographs of epididymal fat tissues from AIM-felinized, WT, AIM −/− , and Δsµ mice fed an HFD for 12 weeks stained with H E. Adipocyte sizes of 50 independent adipocytes in different areas were evaluated. Results are presented as average ± SEM (in μm 2 ). Scale bars, 100 μm. ( d ) Representative photomicrographs of epididymal fat tissues from AIM-felinized, WT, and AIM −/− mice (fed an HFD for 12 weeks) stained for AIM (blue), F4/80 (macrophage marker; green), and IgM (red, WT and AIM −/− mice. See Supplementary Fig. 1 for AIM-felinized mice). Yellow arrows represent where IgM-free AIM signals exist, while red arrows indicate where AIM is co-stained with IgM. Scale bars, 100 μm. (e) The mRNA levels of AIM and F4/80 were assessed by qPCR using RNA isolated from epididymal fat in WT mice before or after being fed an HFD for 12 weeks. Values were normalized to those of GAPDH and presented as the expression relative to that of AIM from lean WT mice liver and of F4/80 from fat tissues before being fed an HFD (n = 4 per group). Error bar indicates the SEM. (f) Representative photomicrographs of liver from AIM-felinized, WT, AIM −/− , and Δsµ mice fed an HFD for 12 weeks stained with H E. Scale bars, 100 µm. (g) Liver weights and TG contents from AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 6–9 for 12-week HFD per group). Error bar indicates the SEM.

    Article Snippet: Primary antibodies: AIM (rab2 rabbit polyclonal for mice AIM in western blotting and immunohistochemistry, PAC-11 rabbit polyclonal for feline AIM in western blotting, and clone #33 mouse monoclonal for feline AIM in western blotting and immunohistochemistry; established in our laboratory), IgM (A21044, goat polyclonal, Alexa 594 conjugated, Thermo Fisher Scientific, Waltham, MA), F4/80 (clone: BM8 for immunohistochemistry; Thermo Fisher Scientific), CD36 (clone: MF3 for immunohistochemistry; Abcam, Cambridge, UK) and gp73 (goat polyclonal for immunohistochemistry; Santa Cruz, Dallas, TX).

    Techniques: Mouse Assay, Staining, Marker, Real-time Polymerase Chain Reaction, Isolation, Expressing