Journal: EBioMedicine

Article Title: Immune-infiltration based signature as a novel prognostic biomarker in gastrointestinal stromal tumour

doi: 10.1016/j.ebiom.2020.102850

Figure Lengend Snippet: Kaplan-Meier analysis of RFS according to individual TIL populations in the TC. (a) CD3 + TC. (b) CD8 + TC. (c) CD45RO + TC. (d) Foxp3 + TC. (e) CD4 + TC. (f) NKp46 + TC. (g) CD20 + TC.

Article Snippet: Then, sections were incubated with primary antibodies against CD3 (Ventana, 790–4341; ready-to-use), CD8 (Dako, M7103; 1:100 dilution), CD45RO (Origene, TA807197; 1:100 dilution), CD4 (Abcam, ab213215; 1:300 dilution), Foxp3 (Abcam, ab22510; 1:200 dilution), NKp46 (Abcam, ab214468; 1:500 dilution), CD20 (Abcam, ab27093; 1:200 dilution), programmed death-1(PD-1) (Cell signaling Technology, 43,248; 1:50 dilution), and programmed death-ligand 1 (PD-L1) (Dako, M3653; 1:50 dilution) at 4 °C overnight.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Sirtuin-1 (SIRT1) Is Required for Promoting Chondrogenic Differentiation of Mesenchymal Stem Cells

doi: 10.1074/jbc.M114.568790

Figure Lengend Snippet: Characterization of MSCs. A, in monolayer culture the human MSCs (panel a) showed a polymorphic, fibroblast-like morphology. The MSCs expressed positive stem cell-specific markers CD90+ (panel b) and CD105+ (panel c) and were negative for the hematopoietic stem cell markers CD45− (panel d) and CD34− (panel e). Magnification: ×10 (panel a), ×40 (panels b–e), and bar, 1 μm. B, to quantify the stem cell marker-positive cells, the labeled cultures were examined by counting 300 cells from 10 microscopic fields. The examination was performed in triplicate, and the results are provided as the mean values with S.D. from three independent experiments. Values were compared with the control, and statistically significant values with p < 0.05 are designated by a star, and p < 0.01 is designated by two stars. Co., without primary antibody, followed by incubation with rhodamine-coupled secondary antibodies and counterstaining with DAPI to visualize the cell nuclei.

Article Snippet: Monoclonal anti-Sox9, monoclonal anti-CD105, monoclonal anti-CD90, monoclonal anti-CD45, and monoclonal anti-CD34 were purchased from Acris Antibodies GmbH (Hiddenhausen, Germany).

Techniques: Marker, Labeling, Incubation

Journal: Cells

Article Title: Short-Term Evaluation of Cellular Fate in an Ovine Bone Formation Model

doi: 10.3390/cells10071776

Figure Lengend Snippet: Details of the antibodies used for immunocytochemistry.

Article Snippet: Anti-CD45 (10 μg/mL) , WS0544B-100 (Kingfisher Biotech) , Invitrogen, A21236 (10 μg/mL).

Techniques: Immunocytochemistry

Journal: Journal of Diabetes Research

Article Title: Renal Protection by Genetic Deletion of the Atypical Chemokine Receptor ACKR2 in Diabetic OVE Mice

doi: 10.1155/2016/5362506

Figure Lengend Snippet: Knockout of the ACKR2 gene reduces leukocyte infiltration in diabetic mice. (a) Representative images of CD45 staining, original magnification 200x. (b) Quantitative analysis of leukocyte infiltration scored as CD45 positive pixel area per visual field. Twenty-four random fields from 3 mice per group were measured. ∗ indicates p < 0.05 versus OVE. Statistical comparisons were performed by one-way ANOVA.

Article Snippet: Renal inflammatory cell infiltration was evaluated by staining sections with rat anti-mouse CD45 antibody (Angio-Proteomie, Boston, MA).

Techniques: Knock-Out, Staining

Journal: JCI Insight

Article Title: Inhibition of B cell–dependent lymphoid follicle formation prevents lymphocytic bronchiolitis after lung transplantation

doi: 10.1172/jci.insight.123971

Figure Lengend Snippet: Left lungs from C57BL/6J (B6) and HLA-A2–knockin (HLA) mice on a B6 background (HLA) were orthotopically transplanted into B6 recipient mice and analyzed 2 months after LTx (B6→B6, n = 4, HLA→B6, n = 4). (A) Representative immunofluorescence staining: 2 months after LTx, lungs of the indicated mice were stained for the T cell marker CD3 (green), the B cell marker CD45R (83), and counterstained with the nuclear marker DAPI. CD3+ and CD19+ cells infiltrating the grafts, analyzed by flow cytometry (FACS), 2 months after LTx. Representative FACS plots and quantification of CD3+ and CD19+ cells, as percentage of live cells, are shown. Data are expressed as mean ± SEM and were analyzed with a Mann-Whitney test; *P < 0.05. (B) Representative images of double immunofluorescence staining for the club cell marker CC10 (green) and the B cell marker CD45R (83) of the indicated mice, 2 months after LTx. Scale bars: 100 μm. (C) Representative images of double immunofluorescence staining for the club cell marker CC10 (green) and the B cell marker CD20 (83) of a lymphocytic bronchiolitis (LB) lesion from human BOS explant. (D) Flow cytometry analysis of the B cell activation markers CD69, MHCII, CD80, and IgG. Left: Representative FACS plots of the above-listed surface markers, gated on CD19+ cells, of the indicated mice. Right: Quantification of the indicated cell populations, expressed as percentage of CD19+ cells. Data are expressed as mean ± SEM and were analyzed with a Mann-Whitney test; *P < 0.05. (E) Representative images of double immunofluorescence staining for IgM (83) and IgG (green) of the indicated mice, 2 months after LTx. Indicated separated fluorescence channels and merged images are presented. Scale bars: 100 μm. Br, bronchus.

Article Snippet: The primary antibodies and dilutions were used as follows: anti-CC10 (clone E-11, Santa Cruz), anti-acetylated tubulin (clone Acteyl-K40, Abcam), anti-CD3 (clone SP7, Abcam), anti-CD45R (clone RA3-6B2, Labome), anti-EBI2 (anti-GPR183/EBI2 rabbit polyclonal [C terminus] antibody, catalog TA316825, Origene), anti-CD138 (catalog PA5-16918, Thermo Fisher Scientific), anti–HLA-A (clone EP1395Y, Abcam).

Techniques: Knock-In, Immunofluorescence, Staining, Marker, Flow Cytometry, MANN-WHITNEY, Double Immunofluorescence Staining, Activation Assay, Fluorescence

Journal: JCI Insight

Article Title: Inhibition of B cell–dependent lymphoid follicle formation prevents lymphocytic bronchiolitis after lung transplantation

doi: 10.1172/jci.insight.123971

Figure Lengend Snippet: Left lungs from HLA-A2–knockin mice on a B6 background (HLA) were orthotopically transplanted into μMT+/– control or μMT–/– recipient mice, both on a B6 background. The mice were analyzed 2 months after orthotopic left lung transplantation (LTx) (HLA→μMT+/–, n = 4, HLA→μMT–/–, n = 4). (A) Lungs from indicated mice acquired with the x-ray dark-field imaging technique. Arrows point toward the left lung graft. (B) Representative scans (original magnification, ×2; scale bars: 1000 μm) and zoomed bronchi (original magnification, ×20; scale bars: 100 μm) from indicated transplanted mice, stained with Masson’s trichrome. (C) Quantification of the epithelial area and peribronchial area of the indicated mice. Data are expressed as mean ± SEM of all the quantified bronchi and were analyzed with a 2-way ANOVA with a Bonferroni post-test; ***P < 0.001. (D) Double immunofluorescence for CC10+ club cells (83) and AcTUB+ ciliated cells (green) of the indicated mice, 2 months after LTx. Scale bars: 100 μm. Br, bronchus; V, vessel. Quantification of the CC10+ and AcTUB+ cells, expressed as mm of bronchial basement membrane. Data are expressed as mean ± SEM of all the quantified bronchi and were analyzed with a Mann-Whitney test; ***P < 0.001. (E) Mediastinal lymph nodes (MLN) were mechanically dissociated, stimulated ex vivo with anti-CD3/CD28 (for T helper responses) or anti-CD40+LPS (for B effector responses), and analyzed by FACS after surface or surface and intracellular staining. Top left: CD19+ B cells. The data were analyzed with a 1-way ANOVA with a Kruskal-Wallis post-test; **P < 0.01. Top right: CD19+ B cells producing IFN-γ, IL-4, and IL-17A. Data were analyzed with a 2-way ANOVA followed by a Bonferroni post-test; ***P < 0.001. Bottom left: CD4+ T cells. The data were analyzed with a 1-way ANOVA with a Kruskal-Wallis post-test; * P < 0.05. Bottom right: CD4+ T cells producing IFN-γ, IL-4, and IL-17A (respectively reflecting Th1, Th2, and Th17 populations). The data were analyzed with a 2-way ANOVA with a Bonferroni post-test; ***P < 0.001. (F) Double immunofluorescence for CD3+ T cells (green) and CD45R+ B cells (83) of the indicated mice. Scale bars: 100 μm. Quantification of the follicle areas from indicated mice. Data are expressed as mean ± SEM and were analyzed with a Mann-Whitney test; ***P < 0.001.

Article Snippet: The primary antibodies and dilutions were used as follows: anti-CC10 (clone E-11, Santa Cruz), anti-acetylated tubulin (clone Acteyl-K40, Abcam), anti-CD3 (clone SP7, Abcam), anti-CD45R (clone RA3-6B2, Labome), anti-EBI2 (anti-GPR183/EBI2 rabbit polyclonal [C terminus] antibody, catalog TA316825, Origene), anti-CD138 (catalog PA5-16918, Thermo Fisher Scientific), anti–HLA-A (clone EP1395Y, Abcam).

Techniques: Knock-In, Transplantation Assay, Imaging, Staining, Immunofluorescence, MANN-WHITNEY, Ex Vivo

Journal: JCI Insight

Article Title: Inhibition of B cell–dependent lymphoid follicle formation prevents lymphocytic bronchiolitis after lung transplantation

doi: 10.1172/jci.insight.123971

Figure Lengend Snippet: (A) Left lungs from C57BL/6J (B6) and HLA-A2–knockin (HLA) mice on a B6 background (HLA) were removed from the donors, cuffed, and orthotopically transplanted into B6 recipient mice. The mice were analyzed 2 months after LTx. Representative double immunofluorescence stainings for the B cell marker CD45R (green) and EBI2 (83): single channel and merged images. (B) Representative double immunofluorescence stainings of human bronchiolitis obliterans syndrome (BOS) explants for the T cell marker CD3 (top, green), the B cell marker CD20 (top, red), the germinal center marker GL7 (bottom, green), and EBI2 (bottom, red). Single channel and merged images. Scale bars: 100 μm.

Article Snippet: The primary antibodies and dilutions were used as follows: anti-CC10 (clone E-11, Santa Cruz), anti-acetylated tubulin (clone Acteyl-K40, Abcam), anti-CD3 (clone SP7, Abcam), anti-CD45R (clone RA3-6B2, Labome), anti-EBI2 (anti-GPR183/EBI2 rabbit polyclonal [C terminus] antibody, catalog TA316825, Origene), anti-CD138 (catalog PA5-16918, Thermo Fisher Scientific), anti–HLA-A (clone EP1395Y, Abcam).

Techniques: Knock-In, Immunofluorescence, Marker

Journal: JCI Insight

Article Title: Inhibition of B cell–dependent lymphoid follicle formation prevents lymphocytic bronchiolitis after lung transplantation

doi: 10.1172/jci.insight.123971

Figure Lengend Snippet: (A and B) Left lungs from HLA-A2–knockin mice on a B6 background (HLA) were orthotopically transplanted into B6 recipient mice or Ebi2–/– mice on a B6 background. The mice were analyzed 2 months after LTx (HLA→B6, n = 4, HLA→Ebi2–/–, n = 4). (A) Scans showing the distribution of follicles (CD3: green channel, CD45R: red channel, DAPI: blue channel) inside the lung allografts. (B) Left: Quantification of the follicle areas from indicated mice. Data are expressed as mean ± SEM and were analyzed with a Mann-Whitney test; ***P < 0.001. Right: Percentage of follicles per graft section plotted by follicle size. Data are expressed as mean ± SEM and were analyzed with a 2-way ANOVA test, followed by a Bonferroni post-test; ***P < 0.001. (C–F) Left lungs from HLA-A2–knockin mice on a B6 background (HLA) were orthotopically transplanted into B6 or Ebi2–/– recipient mice, both on a B6 background. In one group, the transplanted mice were treated a pharmacological inhibitor of EBI2 (NIBR189, 1 mg/kg, 2 times per week from the first day after LTx). The mice were analyzed 2 months after orthotopic left lung transplantation (LTx) (HLA→B6, n = 3, HLA→Ebi2–/–, n = 4, HLA→B6+NIBR189, n = 3). (C) Scans (original magnification, ×2; scale bars: 1000 μm) and zoomed bronchi (original magnification, ×20; scale bars: 100 μm) from indicated transplanted mice, stained with Masson’s trichrome. (D) Epithelial area and peribronchial areas of the indicated mice. Data are expressed as mean ± SEM of all the quantified bronchi and were analyzed with a 1-way ANOVA with a Kruskal-Wallis post-test; **P < 0.01; ***P < 0.001. (E) Double immunofluorescence and quantification of CC10+ club cells and AcTUB+ ciliated cells of the indicated mice. Scale bars: 100 μm. Data are expressed as mean ± SEM of all the quantified bronchi and were analyzed with a Mann-Whitney test; ***P < 0.001. (F) Allograft resistance and compliance. Data are expressed as mean ± SEM.

Article Snippet: The primary antibodies and dilutions were used as follows: anti-CC10 (clone E-11, Santa Cruz), anti-acetylated tubulin (clone Acteyl-K40, Abcam), anti-CD3 (clone SP7, Abcam), anti-CD45R (clone RA3-6B2, Labome), anti-EBI2 (anti-GPR183/EBI2 rabbit polyclonal [C terminus] antibody, catalog TA316825, Origene), anti-CD138 (catalog PA5-16918, Thermo Fisher Scientific), anti–HLA-A (clone EP1395Y, Abcam).

Techniques: Knock-In, MANN-WHITNEY, Transplantation Assay, Staining, Immunofluorescence

Journal: Neurobiology of disease

Article Title: Characterization and preclinical evaluation of the cGMP grade DNA based vaccine, AV-1959D to enter the first-in-human clinical trials

doi: 10.1016/j.nbd.2020.104823

Figure Lengend Snippet: No increased infiltration of T and B cells was observed in AV-1959D/EP vaccinated mice compared with control animals 2 and 14 days after the last immunization. Table represents average number ± SEM of the infiltrating lymphocytes per brain section. Photomicrographs of the brain sections from immunized ( n = 8/gender/time point) and control ( n = 7/gender/timepoint) mice stained with anti-CD3 and anti-B220. High-power views are taken from the circled areas: S1 (primary somatosensory cortex), RS (retrosplenial cortex), Th (thalamic nucleus), DG (dentate gyrus), Hb (habenular nucleus). Scale bars: low magnification = 2.5 mm, enlarged images =100 μm.

Article Snippet: The levels of microgliosis were identified using Iba1 Rabbit polyclonal antibody (GTX100042, GeneTex) at 1:500 dilution utilizing 0.1 M citrate buffer pretreatment; for detection of mouse astrocytes we used Rabbit polyclonal Glial Fibrillary Acidic Protein (GFAP, RB-087-A, Thermo Fisher Scientific) at 1:500; for analyzing possible T cell infiltration, we utilized Rabbit monoclonal [SP7] antibody to CD3 (ab16669, Abcam) at 1:500; for detection of B-cells, we used Rabbit recombinant monoclonal purified Ab B220 [BL-178–12C7] (A700–012, Bethyl Laboratories) at 1:250, with a 0.1 M citrate buffer pretreatment.

Techniques: Control, Staining

Journal: The Journal of Clinical Investigation

Article Title: Anti-CD45RC antibody immunotherapy prevents and treats experimental autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy syndrome

doi: 10.1172/JCI156507

Figure Lengend Snippet: ( A ) Schematic of the protocol of administration of isotype control or anti-CD45RC mAbs as prevention in 3-week-old Aire –/– rats. ( B ) Representative photographs of Aire –/– rats after 4 months of treatment with either isotype control ( n = 13) or anti-CD45RC mAbs ( n = 13). Arrows indicate the alopecia- and vitiligo-like manifestations. Scale bars: 5 cm. ( C ) Evolution of weight gain in Aire –/– male rats during treatment with isotype control ( n = 6) or anti-CD45RC mAbs ( n = 6) until sacrifice. ANOVA comparing curves: *** P < 0.001. ( D ) Representative pictures of H&E histological analysis of organs from Aire –/– rats at the end of the 4-month treatment with isotype control ( n = 7) or anti-CD45RC mAbs ( n = 7). Black arrows indicate lesions and mononuclear cell infiltrates. Black bars indicate the marginal zone. Scale bars: 250 nm (thymus and spleen), 500 μm (skin and kidney), and 1 mm (pancreas and lung).

Article Snippet: The slides were immersed in 3% Tris–goat serum solution to block nonspecific binding, and then 1:200 CD45RC (clone MT2, catalog AM39022PU-N, Origene) antibody was applied at room temperature and incubated for 1 hour.

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: Anti-CD45RC antibody immunotherapy prevents and treats experimental autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy syndrome

doi: 10.1172/JCI156507

Figure Lengend Snippet: ( A ) Tissue sections of organs from IgM –/– rats were incubated with sera of Aire –/– animals at 4 months of treatment with anti-CD45RC or isotype control mAb. Autoantibodies are depicted in yellow and DAPI in blue. Original magnification, ×20. Images are representative of 3 different experiments. ( B ) Sera from anti-CD45RC or isotype control mAb–treated Aire –/– rats at 4 months of treatment were incubated on Western blot membranes after migration and transfer of tissue-specific self-antigens from IgM –/– rats. Binding of autoantibodies was revealed using an anti-rat IgG biotin-coupled antibody and avidin peroxidase. β-Actin was used as a loading control. Data are representative of 5 different experiments. MLN, mesenteric lymph nodes. ( C ) Anti-cytokine and tissue-specific autoantibodies were quantified by LIPS assay using sera from anti-CD45RC or isotype control mAb–treated Aire –/– rats at 4 months of treatment. Normalization was achieved by division of the obtained value by the mean values obtained from WT animals. Mann-Whitney analysis showed no significant difference between the 2 groups.

Article Snippet: The slides were immersed in 3% Tris–goat serum solution to block nonspecific binding, and then 1:200 CD45RC (clone MT2, catalog AM39022PU-N, Origene) antibody was applied at room temperature and incubated for 1 hour.

Techniques: Incubation, Western Blot, Migration, Binding Assay, Avidin-Biotin Assay, Lips Assay, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: Anti-CD45RC antibody immunotherapy prevents and treats experimental autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy syndrome

doi: 10.1172/JCI156507

Figure Lengend Snippet: ( A – C ) Blood at 2 weeks of treatment ( A ) and spleen ( B ) and thymus ( C ) at 4 months of treatment of Aire –/– rats with isotype control or anti-CD45RC mAbs were stained for the expression of CD45RC on CD8 + and CD4 + T cells by flow cytometry and compared with those from Aire +/+ rats. ( A ) Shown is a representative staining of 4–7 animals. The gates indicate the high, low, and negative subsets of CD45RC. Mean ± SEM of CD45RC expression on CD4 + and CD8 + T cells after 2 weeks of treatment is summarized in the graphs on the right. ANOVA: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( B and C ) Results are shown as mean ± SEM of CD45RC subsets among CD4 + T cells (left) or CD8 + T cells (right). ANOVA: * P < 0.05, ** P < 0.01, *** P < 0.001. ( D ) Cell subset distribution was analyzed by flow cytometry among immune cells from spleen, mesenteric lymph nodes (MLN), and thymus of untreated Aire +/+ and isotype control or anti-CD45RC mAb–treated Aire –/– rats at the time of sacrifice. Results are shown as mean ± SEM. ANOVA: *** P < 0.001, **** P < 0.0001.

Article Snippet: The slides were immersed in 3% Tris–goat serum solution to block nonspecific binding, and then 1:200 CD45RC (clone MT2, catalog AM39022PU-N, Origene) antibody was applied at room temperature and incubated for 1 hour.

Techniques: Staining, Expressing, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: Anti-CD45RC antibody immunotherapy prevents and treats experimental autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy syndrome

doi: 10.1172/JCI156507

Figure Lengend Snippet: ( A ) Proportion of B cell subtypes in the spleen of Aire –/– rats after 4 months of treatment with the anti-CD45RC or isotype control mAb or WT untreated rats. ( B ) B cells from Aire +/+ rats were stimulated in vitro for 48 hours with CpG ODN 1668, anti-CD40, and anti-IgM mAbs in the presence of anti-CD45RC or isotype control mAbs. Maturation of B cells was analyzed by flow cytometry quantification of each B cell subpopulation. ANOVA: * P < 0.05. ( C ) After 48 hours of in vitro stimulation, B cells from Aire +/+ and Aire –/– rats were stained for flow cytometry to study their viability and expression of CD45RC and activation markers such as CD80, CD40, and MHC class II (RT1-B). Data from each experiment were normalized to the mean value from all the experiments. Results of multiple t test statistical analysis comparing isotype control with anti-CD45RC mAb conditions in Aire –/– or Aire +/+ B cells. Multiple t test: * P < 0.05, ** P < 0.01, *** P < 0.001. ( D ) IgM and IgG ELISA quantification in the culture supernatant of B cells stimulated for 48 hours. Multiple t test: * P < 0.05. ( E ) Ratio of IgM versus IgG production in culture supernatant of B cells stimulated for 48 hours.

Article Snippet: The slides were immersed in 3% Tris–goat serum solution to block nonspecific binding, and then 1:200 CD45RC (clone MT2, catalog AM39022PU-N, Origene) antibody was applied at room temperature and incubated for 1 hour.

Techniques: In Vitro, Flow Cytometry, Staining, Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Clinical Investigation

Article Title: Anti-CD45RC antibody immunotherapy prevents and treats experimental autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy syndrome

doi: 10.1172/JCI156507

Figure Lengend Snippet: ( A ) Ratio of CD4 + CD25 + CD127 lo/– or CD8 + CD45RC lo/– Tregs versus CD45RC hi Tconv cells in Aire –/– rats treated with isotype control ( n = 7) versus anti-CD45RC mAb ( n = 7). ANOVA: * P < 0.05. ( B ) Matrix showing the number of genes differentially expressed between CD4 + (left panel) and CD8 + (right panel) Tregs from the following groups: WT rats ( n = 8) and Aire –/– rats treated with the isotype control ( n = 5) or the anti-CD45RC mAb ( n = 5). ( C ) DGE RNA sequencing heatmap analysis of CD8 + CD45RC lo Tregs showing the relative expression of genes. Columns correspond to samples, and rows correspond to differentially expressed genes. Expression values were averaged per sample and scaled per gene. Blue represents lowly-expressed genes and red represents highly expressed genes. ( D ) Normalized enrichment score of biological pathways upregulated or downregulated in Aire –/– rats treated with the anti-CD45RC mAb ( n = 5) compared with Aire –/– rats treated with the isotype control mAb ( n = 5).

Article Snippet: The slides were immersed in 3% Tris–goat serum solution to block nonspecific binding, and then 1:200 CD45RC (clone MT2, catalog AM39022PU-N, Origene) antibody was applied at room temperature and incubated for 1 hour.

Techniques: RNA Sequencing Assay, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Anti-CD45RC antibody immunotherapy prevents and treats experimental autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy syndrome

doi: 10.1172/JCI156507

Figure Lengend Snippet: ( A ) Schematic of the protocol of treatment in the curative setting of 3-month-old Aire –/– rats with isotype control or anti-CD45RC mAbs. ( B ) Representative photographs of Aire –/– rats after 4 months of treatment with either isotype control ( n = 8) or anti-CD45RC mAbs ( n = 11). Arrows depict alopecia. Scale bars: 5 cm. ( C ) Evolution of weight gain in male Aire –/– rats treated with isotype control ( n = 4) or anti-CD45RC mAbs ( n = 5) until sacrifice. ANOVA analysis showed no significant difference between the 2 groups. ( D ) Representative pictures of H&E histological analysis of organs from Aire –/– rats at the end of the treatment with isotype control ( n = 4) or anti-CD45RC mAbs ( n = 5). Black arrows indicate autoimmune lesions and immune cell infiltrates. Black bars indicate the marginal zone. Scale bars: 250 nm (spleen, lung, and kidney), 500 μm (skin and pancreas), and 1 mm (thymus).

Article Snippet: The slides were immersed in 3% Tris–goat serum solution to block nonspecific binding, and then 1:200 CD45RC (clone MT2, catalog AM39022PU-N, Origene) antibody was applied at room temperature and incubated for 1 hour.

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: Anti-CD45RC antibody immunotherapy prevents and treats experimental autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy syndrome

doi: 10.1172/JCI156507

Figure Lengend Snippet: ( A ) Organ serial sections from the same IgM –/– rats as shown in were incubated with different sera from anti-CD45RC or isotype mAb–treated Aire –/– animals at 4 months of treatment as indicated. Autoantibodies are depicted in yellow and DAPI in blue. Original magnification, ×20. Images are representative of 3 animals per group. ( B ) Sera from anti-CD45RC or isotype mAb–treated Aire –/– rats at 4 months of treatment were incubated on Western blot membranes after migration and transfer of tissue-specific self-antigens from IgM –/– rats. β-Actin was used as a loading control. Data are representative of 5 animals per group. MLN, mesenteric lymph nodes. ( C ) Anti-cytokine and -Pdia2 autoantibodies were quantified by LIPS assay using sera from anti-CD45RC or isotype mAb–treated Aire –/– rats at 4 months of treatment ( n = 5 each). Normalization was achieved by division of the obtained value by the mean values obtained from WT animals. Mann-Whitney: * P < 0.05, ** P < 0.01.

Article Snippet: The slides were immersed in 3% Tris–goat serum solution to block nonspecific binding, and then 1:200 CD45RC (clone MT2, catalog AM39022PU-N, Origene) antibody was applied at room temperature and incubated for 1 hour.

Techniques: Incubation, Western Blot, Migration, Lips Assay, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: Anti-CD45RC antibody immunotherapy prevents and treats experimental autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy syndrome

doi: 10.1172/JCI156507

Figure Lengend Snippet: ( A ) PBMCs of APECED patients ( n = 11) and healthy donors ( n = 16) were stained for flow cytometry analysis showing the expression of CD45RC on CD4 + (left) and CD8 + (right) T cells; t test: * P < 0.05, ** P < 0.01, *** P < 0.001. ( B ) Expression of FOXP3 and CD45RC on CD4 + (top line) and CD8 + T cells (bottom line) from APECED patients and healthy donors. ( C ) Ratio of FOXP3 + Tregs versus CD45RC hi Tconv cells in healthy donors ( n = 16) versus APECED patients ( n = 11); t test: * P < 0.05. ( D ) Expression of IL-10, IL-34, CD40L, Tbet, CD103, CD127, and PD-1 by CD4 + and CD8 + T cells from healthy donors and APECED patients; t test: * P < 0.05, ** P < 0.01, *** P < 0.001. ( E ) Representative immunohistochemical staining of CD45RC with an anti–human CD45RC mAb in stomach and small intestine paraffin-embedded tissue from 2 APECED patients with autoimmune gastritis and enteropathy (arrows) compared with stomach tissue of an APECED patient without autoimmune gastritis. Non-APECED human tonsil biopsy tissue was used as positive control. ( F ) Proportion of apoptotic CD45RA hi T cells induced after a 3-hour in vitro incubation of PBMCs, from healthy donors ( n = 6) or APECED patients ( n = 6) with the anti-CD45RC or isotype control mAbs. One-way ANOVA repeated measures, Bonferroni’s post hoc test: *** P < 0.001, **** P < 0.0001.

Article Snippet: The slides were immersed in 3% Tris–goat serum solution to block nonspecific binding, and then 1:200 CD45RC (clone MT2, catalog AM39022PU-N, Origene) antibody was applied at room temperature and incubated for 1 hour.

Techniques: Staining, Flow Cytometry, Expressing, Immunohistochemical staining, Positive Control, In Vitro, Incubation

Journal: The American Journal of Pathology

Article Title: ABIN1 Determines Severity of Glomerulonephritis via Activation of Intrinsic Glomerular Inflammation

doi: 10.1016/j.ajpath.2017.08.018

Figure Lengend Snippet: ABIN1[D485N] mice have enhanced glomerular inflammation after nephrotoxic sera (NTS) administration. A: Immunohistochemistry (IHC) analysis was used to compare nuclear expression of the NF-κB p65 subunit (indicative of NF-κB activation) within the glomerulus at 2 hours after NTS or normal serum (NS) control administration in wild-type (WT) and ABIN1[D485N] mice. B: Representative images (arrows indicate examples of positive nuclear p65 expression). C: Isolated glomeruli from WT or ABIN1[D485N] kidneys revealed marked transcriptional changes in a number of proinflammatory cytokines, chemokines, and adhesion molecules. IHC was used to reveal increased levels of CD45-positive and myeloperoxidase (MPO)–positive cells within the glomerulus at 2 hours (D) and 24 hours (E) after NTS. F: Representative images of glomeruli 24 hours after NTS (arrows indicate location of granular MPO staining). Error bars represent SEM, and the t-test was used to compare values between 2 groups. n = 3 per condition with 10 glomeruli counted per sample (A); n = 4 per condition (C); n = 5 per condition with 30 glomeruli counted per sample (D and E). ∗P < 0.05, ∗∗P < 0.01 versus controls. Scale bar = 50 μm (B and F). CCL, chemokine (C-C motif) ligand; EGF, epidermal growth factor; ICAM-1, intercellular adhesion molecule-1; IFN, interferon; MMP-7, matrix metalloproteinase-7; MYH9, myosin heavy chain-9; RELa, NF-kB p65 subunit; TGF-β1, transforming growth factor-β1; TNF-α, tumor necrosis factor-α; VCAM-1, vascular cell adhesion molecule-1.

Article Snippet: 24 Primary antibodies dilutions were applied at 1:200 for WT-1 (Santa Cruz Biotechnology, Dallas, TX) and CD45 (Angio-Proteomie, Boston, MA), NF-κB (p65) (Santa Cruz Biotechnology) and 1:50 for myeloperoxidase (MPO) (AbCam, Cambridge, MA) and fluorescein isothiocyanate conjugated anti-C3 (PA1-29718; Thermo Fisher Scientific).

Techniques: Immunohistochemistry, Expressing, Activation Assay, Isolation, Staining

Journal: Journal of Veterinary Science

Article Title: Comparison of the characteristics of canine adipose tissue-derived mesenchymal stem cells extracted from different sites and at different passage numbers

doi: 10.4142/jvs.2018.19.1.13

Figure Lengend Snippet: Expressions of CD44, CD90, and CD45 on adipose tissue-derived mesenchymal stem cells of subcutaneous (Sc) or visceral (Vs) origin were assessed by performing flow cytometry (n = 2). Data are representative of analyzed passages.

Article Snippet: Then, cells were immunostained for 30 min at 4℃ in the dark with 10 µL of each of allophycocyanin-conjugated anti-CD44 (clone IM7; Leinco Technologies, USA) and fluorescein isothiocyanate-conjugated anti-CD45/LCA (CD45RO, clone UCHL1; Acris Antibodies, USA) antibodies, as well as 20 µL of phycoerythrin-conjugated anti-CD90 antibody (THY1, clone 5E10; antibodies-online, USA).

Techniques: Derivative Assay, Flow Cytometry