rat anti mouse madcam 1 antibody  (SouthernBiotech)

Journal: PLoS ONE

Article Title: Traceless Bioresponsive Shielding of Adenovirus Hexon with HPMA Copolymers Maintains Transduction Capacity In Vitro and In Vivo

doi: 10.1371/journal.pone.0082716

Figure Lengend Snippet: To analyze whether HPMA copolymer shielding altered the cell type in the spleen that was transduced after vector injection we performed immunohistological staining of histological cryosections against CD11c (A), ER-TR7 (B) and MADCAM-1 (C). Female BALB/c were injected with HPMA copolymer-shielded EGFP-expressing Ad vector particles. 72 h later spleens were harvested and analyzed by histological cryosections. Representative sections are shown. Several cell types in the spleen appear to be transduced by Ad mainly in the marginal zone. Shielding with HPMA copolymers reduced the overall transduction but did not seem to alter the transduction pattern or type of transduced cell. Representative sections are shown. HPMA copolymers # 268, # 279, # 280 and # 283 not shown. Abbreviations: HexCys: unshielded AdHexCys, the “+ Polymer-number” indicates a shielding of AdHexCys with the respective HPMA copolymer, neut.: neutral (uncharged), pos.: positively charged, irrev.: irreversible (mal-)shielding, rev.: bioresponsive (PySS-)shielding.

Article Snippet: Rat anti-mouse ER-TR7 mAB (1∶33; Abcam, ab51824, Cambridge, GB) and Rat anti-mouse MADCAM-1 mAB (1∶33; eBioscience, Inc., 14-5997-85, San Diego, CA, USA) were used with the secondary AB goat anti-rat AlexaFluor555 (1∶500; Invitrogen Ltd., Paisley, GB).

Techniques: Plasmid Preparation, Injection, Staining, Expressing, Transduction

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: MAdCAM-1/α4β7 Integrin-Mediated Lymphocyte/Endothelium Interactions Exacerbate Acute Immune-Mediated Hepatitis in Mice

doi: 10.1016/j.jcmgh.2020.12.003

Figure Lengend Snippet: ConA-induced liver damage is ameliorated in MAdCAM-1–deficient and β7 integrin–deficient mice. Plasma and liver specimens were sampled at 8, 20, or 48 hours after ConA administration. The data shown are representative of 3 independent experiments. Data represent mean ± SEM. Statistical significance was calculated in panels A , C , E , F , and G by the Mann-Whitney nonparametric t test (∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001). WT (black bars), β7 integrin Δ/Δ (white bars), and MAdCAM-1 Δ/Δ mice (gray bars). ( A ) Quantification of serum alanine aminotransferase (ALT). WT (n = 9–10), β7 integrin Δ/Δ (n = 5–11), and MAdCAM-1 Δ/Δ mice (n = 7–10). ( B ) Representative photomicrographs of H&E-stained liver sections with marked necrotic areas (original magnification ×10; scale bar = 100 μm) and ( C ) quantification of necrotic area fraction. WT (n = 4), β7 integrin Δ/Δ (n = 4–5), and MAdCAM-1 Δ/Δ mice (n = 3–4). ( D ) Representative photomicrographs (original magnification ×10; scale bar = 100 μm) and ( E ) quantification of bright green apoptotic cells as detected by TUNEL assay 8 or 20 hours post–ConA injection. WT (n = 9–12), β7 integrin Δ/Δ (n = 7–10), and MAdCAM-1 Δ/Δ mice (n = 10), and untreated control mice (n = 9 each). ( F ) Quantification of serum ALT. (WT: n = 7–9, MAdCAM-1 Δ/Δ: n = 9–10). ( G ) Quantification of apoptotic cells as detected by TUNEL assay on liver sections. WT: n = 3–7, MAdCAM-1 Δ/Δ: n = 4–10.

Article Snippet: Sections were then incubated overnight with purified rat anti-mouse MAdCAM-1 (BD Biosciences, Heidelberg, Germany) diluted in 2% BSA in TBST ++ .

Techniques: MANN-WHITNEY, Staining, TUNEL Assay, Injection

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: MAdCAM-1/α4β7 Integrin-Mediated Lymphocyte/Endothelium Interactions Exacerbate Acute Immune-Mediated Hepatitis in Mice

doi: 10.1016/j.jcmgh.2020.12.003

Figure Lengend Snippet: MAdCAM-1– and β7 integrin–deficient mice display reduced clotting following ConA treatment. Plasma and liver specimens were sampled at 8 or 20 hours after ConA administration. ( A ) Representative photomicrographs of H&E-stained liver sections at original magnification ×20. Scale bar = 100 μm. Prominent hemostasis marked by black arrowheads is seen in the sinusoids. ( B ) Quantification of clotting in H&E-stained liver sections shown as percentage of the section area (n ≥ 6 mice per group). Data represent mean ± SEM. Statistical significance was calculated in by the Mann-Whitney nonparametric t test (∗ P < .05, ∗∗ P < .01). WT (black bars), β7 integrin Δ/Δ (white bars), and MAdCAM-1 Δ/Δ mice (gray bars).

Article Snippet: Sections were then incubated overnight with purified rat anti-mouse MAdCAM-1 (BD Biosciences, Heidelberg, Germany) diluted in 2% BSA in TBST ++ .

Techniques: Coagulation, Staining, MANN-WHITNEY

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: MAdCAM-1/α4β7 Integrin-Mediated Lymphocyte/Endothelium Interactions Exacerbate Acute Immune-Mediated Hepatitis in Mice

doi: 10.1016/j.jcmgh.2020.12.003

Figure Lengend Snippet: MAdCAM-1– and β7 integrin–deficient mice display reduced parameters of hemostasis following ConA treatment. Representative pictures of cryostat sections from WT livers 8 hours after ConA administration, which have been stained with ( A ) anti-PAI-1 antibody (green), anti-CD146 antibody (red), and DAPI (blue); ( B ) anti-TF antibody (red), β-catenin (green), and DAPI (blue); or ( C ) anti-CD41 (red), β-catenin (green), and DAPI (blue) and quantification as percentage of the fluorescently stained section area (n = 5 mice per group). Representative photomicrographs at original magnification ×40. Scale bar = 100 μm. ( A ) mRNA levels of Pai - 1 (WT [n = 7], β7 integrin Δ/Δ [n = 5], MAdCAM-1 Δ/Δ mice [n = 4]), and ( B ) Tf (WT [n = 7], β7 integrin Δ/Δ [n = 4], MAdCAM-1 Δ/Δ mice [n = 3]) in liver tissue at 8 hours post–ConA injection. Values are expressed as fold increase over the mean values obtained for healthy control liver tissue from the respective mouse strain. Data show mean ± SD. Statistical significance was calculated by the Mann-Whitney nonparametric t test ∗ P < .05, ∗∗ P < .01; ∗∗∗∗ P < .0001.

Article Snippet: Sections were then incubated overnight with purified rat anti-mouse MAdCAM-1 (BD Biosciences, Heidelberg, Germany) diluted in 2% BSA in TBST ++ .

Techniques: Staining, Injection, MANN-WHITNEY

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: MAdCAM-1/α4β7 Integrin-Mediated Lymphocyte/Endothelium Interactions Exacerbate Acute Immune-Mediated Hepatitis in Mice

doi: 10.1016/j.jcmgh.2020.12.003

Figure Lengend Snippet: MAdCAM-1 expression is induced upon ConA treatment. ( A ) Levels of Madcam-1 mRNA in liver tissue from WT mice at 0, 8, and 20 hours post ConA injection. Control (n = 5), 8 hours post-ConA (n = 7), 20 hours post-ConA (n = 7). mRNA levels are expressed as fold increase over the mean value obtained for healthy control liver tissue. ( A ) Data represent median with interquartile range and are representative of 3 independent experiments. Statistical significance was calculated by the Mann-Whitney nonparametric t test (∗ P < .05, ∗∗ P < .01). Statistical significance of deviation from healthy control animals for each mouse strain is shown as green asterisks above each group. ( B ) Cryostat sections of livers from the indicated mouse strains 8 or 20 hours after ConA administration or without were stained with anti-MAdCAM-1 antibody (green) and DAPI (blue) for visualization of nuclei. In addition, the central section on the right side was stained with anti-CD146 (red). Representative photomicrographs at original magnification ×40. Scale bar = 100 μm.

Article Snippet: Sections were then incubated overnight with purified rat anti-mouse MAdCAM-1 (BD Biosciences, Heidelberg, Germany) diluted in 2% BSA in TBST ++ .

Techniques: Expressing, Injection, MANN-WHITNEY, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: MAdCAM-1/α4β7 Integrin-Mediated Lymphocyte/Endothelium Interactions Exacerbate Acute Immune-Mediated Hepatitis in Mice

doi: 10.1016/j.jcmgh.2020.12.003

Figure Lengend Snippet: MAdCAM-1– or β7 integrin deficiency results in decreased production of ConA-induced proinflammatory mediators. Analysis of the mRNA of pro- and anti-inflammatory mediators in liver tissue from WT, β7 integrin Δ/Δ, and MAdCAM-1 Δ/Δ mice by RT-PCR. Black bars represent data from WT, white bars from β7 integrin Δ/Δ, and gray bars from MAdCAM-1 Δ/Δ mice: Arg ( arginase ), Fizz1 ( found in inflammatory zone 1), Cd38 ( cluster of differentiation 38 ), ( Ifn ( interferon ), Il ( interleukin ), Mcp-1 ( monocyte chemoattractant protein-1 ), Mip-1α ( macrophage inflammatory protein-1α ), iNos ( inducible nitric oxide reductase ), Pparg ( peroxisome proliferator-activated receptor gamma ), Tgf-β ( tumor growth factor-β ), Tnf-α (tumor necrosis factor-α ). ( A ) Analysis in homeostasis. For quantification, values are expressed as delta (Δ) ct values between the genes of interest and the housekeeping gene Gapdh (n = 4–9). Data represent mean ± SD. The statistical significance of variance between different genotypes was calculated by Mann-Whitney nonparametric t test (∗ P < .05). ( B , C ) Analysis 8 hours after ConA injection. For quantification, values are expressed as fold increase over the mean values obtained for control liver tissue from the respective untreated mouse strain. WT mice (n = 12), β7 integrin Δ/Δ mice (n = 10–11), MAdCAM-1 Δ/Δ mice (n = 8–10). Data represent ( B ) mean ± SD and ( C ) mean ± SEM and are representative of 3 independent experiments. Statistical significance in panel B was calculated by the Mann-Whitney nonparametric t test and in panel C by 1-way analysis of variance with Tukey’s multiple comparison posttest. Statistical significance of deviation from healthy control mice for each mouse strain is shown as green asterisks above each group and was calculated by the Mann-Whitney nonparametric t test (∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, ∗∗∗∗ P < .0001).

Article Snippet: Sections were then incubated overnight with purified rat anti-mouse MAdCAM-1 (BD Biosciences, Heidelberg, Germany) diluted in 2% BSA in TBST ++ .

Techniques: Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY, Injection

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: MAdCAM-1/α4β7 Integrin-Mediated Lymphocyte/Endothelium Interactions Exacerbate Acute Immune-Mediated Hepatitis in Mice

doi: 10.1016/j.jcmgh.2020.12.003

Figure Lengend Snippet: Comparative liver analysis of WT, β7 integrin Δ/Δ, and MAdCAM-1 Δ/Δ mice in homeostasis and following ConA treatment. ( A – C ) Total leukocytes were isolated from livers of WT (black bars), β7 integrin Δ/Δ (white bars), and MAdCAM-1 Δ/Δ (gray bars) mice in homeostasis, or ( D , E ) also 8 hours after ConA injection, followed by flow cytometric quantification of leukocytes. Representative FACS (fluorescence-activated cell sorting) dot plots illustrating the gating strategy are shown in Figure 6 . B cells (CD45 + CD3 - CD19 + ), CD4 + T cells (CD45 + CD3 + CD4 + ), CD8 + T cells (CD45 + CD3 + CD8 + ), NKT cells (CD45 + CD3 + NK1.1 + ), Mo-MF (CD45 + CD11b + Ly6G - F4/80 + ), neutrophils (CD45 + CD11b + Ly6G + ), KCs (CD45 + CD11b - Ly6G - F4/80 + ), pDCs (CD45 + CD3 - CD19 - CD11c + PDCA1 + ), cDCs (CD45 + CD3 - CD11c + CD11b + ), macrophages (CD45 + CD11b + F4/80 + ), M1 macrophages (CD45 + CD11b + F4/80 + CD11c + ), and M2 macrophages (CD45 + CD11b + F4/80 + CD206 + ). Cells are shown as percent of CD45 + cells or percent of CD45 + CD11b + F4/80 + cells as indicated. Data represent ( A – C ) mean ± SD and ( D , E ) mean ± SEM and are representative of ( C ) 2 or ( A , B , D , E ) 3 independent experiments. Representative overlay histograms at the right side of panel C show that there is no difference in CD11c and CD206 staining intensity on CD11b + F4/80 + liver macrophages from WT, β7 integrin Δ/Δ, and MAdCAM-1 Δ/Δ mice. Statistical significance was calculated by the Mann-Whitney nonparametric t test and is indicated by the following symbols: ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001. ( A , B ) WT, n = 10; β7 integrin Δ/Δ, n = 10; MAdCAM-1 Δ/Δ, n = 10. ( C ) WT, n = 3; β7 integrin Δ/Δ, n = 3; MAdCAM-1 Δ/Δ, n = 3. ( D ) WT, n = 9–13; β7 integrin Δ/Δ, n = 6–11; MAdCAM-1 Δ/Δ, n = 7–10. ( E ) WT, n = 9–13; β7 integrin Δ/Δ, n = 5–11; MAdCAM-1 Δ/Δ, n = 5–10.

Article Snippet: Sections were then incubated overnight with purified rat anti-mouse MAdCAM-1 (BD Biosciences, Heidelberg, Germany) diluted in 2% BSA in TBST ++ .

Techniques: Isolation, Injection, Fluorescence, FACS, Staining, MANN-WHITNEY

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: MAdCAM-1/α4β7 Integrin-Mediated Lymphocyte/Endothelium Interactions Exacerbate Acute Immune-Mediated Hepatitis in Mice

doi: 10.1016/j.jcmgh.2020.12.003

Figure Lengend Snippet: MAdCAM-1– and β7 integrin–deficient mice display similar levels of ConA-mediated T cell activation. Eight hours after ConA treatment, total leukocytes were isolated from livers and analyzed by flow cytometry. Representative FACS dot plots illustrating the gating strategy are shown in Figure 6 . Data represent ( A ) mean ± SEM and ( C ) mean ± SD and are representative of 3 independent experiments. Statistical significance was calculated by the Mann-Whitney nonparametric t test (∗ P < .05, ∗∗ P < .001, ∗∗∗ P < .001). ( A ) Percentages of activated CD4 + T (CD45 + CD3 + CD4 + CD69 + ), CD8 + T (CD45 + CD3 + CD4 + CD69 + ), and NKT (CD45 + CD3 + NK1.1 + CD69 + ) cells were quantified. WT (black bars, n = 7–9), β7 integrin Δ/Δ (white bars, n = 5–7), and MAdCAM-1 Δ/Δ mice (gray bars, n = 5–7). ( B , C ) ConA-mediated shift in the percentage of β7 integrin–positive cells in WT mice: ( B ) as percent of the maximal cell count for the indicated cells populations and ( C ) as percent of cells expressing β7 integrin within the indicated cell populations. Untreated WT (n = 5) ConA-treated WT (n = 10).

Article Snippet: Sections were then incubated overnight with purified rat anti-mouse MAdCAM-1 (BD Biosciences, Heidelberg, Germany) diluted in 2% BSA in TBST ++ .

Techniques: Activation Assay, Isolation, Flow Cytometry, MANN-WHITNEY, Cell Counting, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: MAdCAM-1/α4β7 Integrin-Mediated Lymphocyte/Endothelium Interactions Exacerbate Acute Immune-Mediated Hepatitis in Mice

doi: 10.1016/j.jcmgh.2020.12.003

Figure Lengend Snippet: Expression of β7 integrin on adaptive and innate immune cells contributes to ConA-mediated liver damage. ( A ) Experimental outline: RAG-2–deficient (RAG-2Δ/Δ), RAG-2/β7 integrin double-deficient (RAG-2Δ/Δ β7Δ/Δ), and RAG-2/MAdCAM-1 double-deficient (RAG-2Δ/Δ MAdCAM-1Δ/Δ) mice were adoptively transferred with WT or β7Δ/Δ cell subsets (lymphocytes, CD4 + and CD8 + T cells, CD8 + T cells, or no cells) and after 10 days subjected to ConA by intravenous tail injection. Six hours later, liver specimens were sampled and analyzed. Black symbols represent RAG-2Δ/Δ, blue symbols represent RAG-2Δ/Δ β7Δ/Δ, and red symbols represent RAG-2Δ/Δ MAdCAM-1Δ/Δ recipient mice. Data are represented as median with interquartile range and statistical significance was calculated by the Mann-Whitney nonparametric t test. Statistical significance of deviation from ConA-treated control mice (without cell transfer) for each mouse strain is shown as green asterisks above each group. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, ∗∗∗∗ P < .0001. ( B ) Quantification of apoptotic cells as detected by TUNEL assay in liver sections (n ≥ 6 mice per group); direct comparison of the indicated recipients. ( C ) Quantification of apoptotic cells as detected by TUNEL assay in liver sections (n ≥ 6 mice per group); direct comparison of the indicated donor cell populations.

Article Snippet: Sections were then incubated overnight with purified rat anti-mouse MAdCAM-1 (BD Biosciences, Heidelberg, Germany) diluted in 2% BSA in TBST ++ .

Techniques: Expressing, Injection, MANN-WHITNEY, TUNEL Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: MAdCAM-1/α4β7 Integrin-Mediated Lymphocyte/Endothelium Interactions Exacerbate Acute Immune-Mediated Hepatitis in Mice

doi: 10.1016/j.jcmgh.2020.12.003

Figure Lengend Snippet: Expression of β7 integrin on adaptive and innate immune cells contributes to ConA-mediated clotting and is crucial for ConA-induced MAdCAM-1 expression. RAG-2–deficient (RAG-2Δ/Δ), RAG-2/β7 integrin double-deficient (RAG-2Δ/Δ β7Δ/Δ), and RAG-2/MAdCAM-1 double-deficient (RAG-2Δ/Δ MAdCAM-1Δ/Δ) mice were adoptively transferred with WT or β7Δ/Δ cell subsets (lymphocytes, CD4 + and CD8 + T cells, CD8 + T cells, or no cells) and after 10 days subjected to ConA by intravenous tail injection. Six hours later, liver specimens were sampled and analyzed. Black symbols represent RAG-2Δ/Δ, blue symbols represent RAG-2Δ/Δ β7Δ/Δ, and red symbols represent RAG-2Δ/Δ MAdCAM-1Δ/Δ recipient mice. Data are represented as median with interquartile range and statistical significance was calculated by the Mann-Whitney nonparametric t test. Statistical significance of deviation from ConA-treated control mice (without cell transfer) for each mouse strain is shown as green asterisks above each group. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, ∗∗∗∗ P < .0001. ( A ) Quantification of clotting in H&E-stained liver sections shown as percentage of the section area (n ≥ 6 mice per group); direct comparison of the indicated recipients. ( B ) Quantification of clotting in H&E-stained liver sections shown as percentage of the section area (n ≥ 6 mice per group); direct comparison of the indicated donor cell populations. ( C ) mRNA levels of Madcam-1 in liver tissue. Values are expressed as fold increase over the mean value obtained for the different ConA-treated recipient mice without cell transfer (n ≥ 6 per group). ( D ) Cryostat sections of livers from ConA-treated RAG-1Δ/Δ or RAG-2Δ/Δβ7Δ/Δ mice, which had been adoptively transferred with WT or β7Δ/Δ lymphocytes as indicated, were stained with anti-MAdCAM-1 antibody (green), anti-CD146 antibody (red), and DAPI (blue) for visualization of nuclei. MAdCAM-1/CD146 double-staining in sinusoids is marked by white arrowheads. Representative photomicrographs at original magnification ×40. Scale bar = 100 μm.

Article Snippet: Sections were then incubated overnight with purified rat anti-mouse MAdCAM-1 (BD Biosciences, Heidelberg, Germany) diluted in 2% BSA in TBST ++ .

Techniques: Expressing, Coagulation, Injection, MANN-WHITNEY, Staining, Double Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: MAdCAM-1/α4β7 Integrin-Mediated Lymphocyte/Endothelium Interactions Exacerbate Acute Immune-Mediated Hepatitis in Mice

doi: 10.1016/j.jcmgh.2020.12.003

Figure Lengend Snippet: Cellular adhesion of lymphocytes in liver sinusoids is reduced in ConA-treated MAdCAM-1–deficient mice. Intravital 2-photon laser scanning microscopy following adoptive transfer of DsRed stained CD45 + cells and ConA injection. ( A ) Flow cytometric analysis of donor lymphocytes from DsRed mice. Lymphocyte preparations were controlled by staining a sample of these cells with an antibody cocktail containing anti-CD45, anti-CD19, and anti-CD3. The figure shows a representative FACS plot (left side) and the quantification of T cells (CD3 + ) and B cells (CD19 + ) (right side), accounting for 95% of the isolated CD45 + cells. ( B – I ) Cells were tracked over a time period of up to 2 hours and up to 3 independent view fields were recorded per animal. Fluorescent cells were detected by automated spot detection. Migration paths and speed of cells were tracked over time in ( B ) WT (CX3CR1 +/eGFP ) and ( C ) MAdCAM-1 Δ/Δ (MAdCAM-1 Δ/Δ/ CX3CR1 +/eGFP ) mice. Fast movement is shown as pink tracks; slow movement is shown as turquoise tracks. Arrows indicate tracks. ( D , E ) Tracks derived from 2-photon laser scanning microscopy imaging shown in panels B and C were plotted using a common origin to depict overall motility of the cells. ( F ) Speed of individual spots and average track speed were analyzed statistically. Results shown are derived from 1 representative animal, calculating migration tracks from at least 3 independent view fields. Spots represent individual cells or tracks. (G) Cellular displacement from the point of origin and total track length were assessed to determine site-specific arrest of cells. ( H ) Statistical assessment of static cells with a track displacement of less than 5 μm. All experiments were performed in groups of 3 animals. Data represent mean ± SD. Statistical significance was calculated by unpaired t test (∗ P < .05; ∗∗∗∗ P < .0001). ( I ) Representative screenshots from 2-photon laser scanning microscopy movies of CX3CR1 +/eGFP control and MAdCAM-1Δ/Δ/CX3CR1 +/eGFP recipient mice, demonstrating that accumulation of DsRed-labeled cells is seen in periportal rather than pericentral areas, independent of the genotype. Scale bar = 50 μM. Donor lymphocytes are seen in red, CX3CR1-positive cells in green.

Article Snippet: Sections were then incubated overnight with purified rat anti-mouse MAdCAM-1 (BD Biosciences, Heidelberg, Germany) diluted in 2% BSA in TBST ++ .

Techniques: Laser-Scanning Microscopy, Adoptive Transfer Assay, Staining, Injection, Isolation, Migration, Derivative Assay, Imaging, Labeling