rat anti meca32  (Novus Biologicals)

Journal: Cell reports

Article Title: Fibroblasts repair blood-brain barrier damage and hemorrhagic brain injury via TIMP2

doi: 10.1016/j.celrep.2022.111709

Figure Lengend Snippet: (A) Schematic illustration of the in vitro ICH model. (B) TEER values of the in vitro ICH model in the presence or absence of fibroblasts. n = 6 biological replicates. ***p = 0.0002 by Student’s t test. (C) Quantification of 4-kDa FITC-dextran leakage in the in vitro ICH model in the presence or absence of fibroblasts. n = 6 biological replicates. **p = 0.0016 and **p = 0.0060 for 30- and 60-min time points by Student’s t test, respectively. (D) Representative images of ZO-1 (green), claudin5 (green), and DAPI (blue) in primary human brain microvascular endothelial cells (HBMECs) under normal conditions (uninjured) and at 48 h after in vitro ICH with or without fibroblasts. Scale bars, 25 μm. (E) Representative images of caveolin-1 (green), meca32 (red), and DAPI (blue) in primary HBMECs under normal conditions (uninjured) and at 48 h after in vitro ICH with or without fibroblasts. Scale bars, 25 μm. (F) Pie chart showing the percentage of each category of proteins relative to total fibroblast-derived proteins identified by LC-MS/MS. (G) TEER values of the in vitro ICH model in the presence of mouse IgG (control), PAI1 function-blocking antibody, and TIMP2 function-blocking antibody. n = 6 biological replicates. **p = 0.0088 by Student’s t test. (H) Quantification of 4-kDa FITC-dextran leakage in the in vitro ICH model in the presence of mouse IgG (control), PAI1 function-blocking antibody, and TIMP2 function-blocking antibody. n = 6 biological replicates, ***p = 0.00013 and ****p < 0.0001 at 30- and 60-min time points by Student’s t test, respectively. (I) Representative western blot image and quantification of TIMP2 expression secreted by fibroblasts after transduction of lentivirus-expressing TIMP2 short hairpin RNA (shRNA) or a scramble sequence (control). n = 5 biological replicates. *p = 0.0121 by Mann-Whitney U test. (J) TEER values of the in vitro ICH model in the presence of control or TIMP2-knockdown fibroblasts. n = 6 biological replicates. **p = 0.0017 by Student’s t test. (K) Quantification of 4-kDa FITC-dextran leakage in the in vitro ICH model in the presence of control or TIMP2-knockdown fibroblasts. n = 6 biological replicates. ****p < 0.0001 by Student’s t test. (L) Schematic illustration of in vitro TIMP2 rescue experiments. (M) TEER values of the in vitro ICH model in the presence of saline (control) or recombinant TIMP2 protein. n = 6 biological replicates. **p = 0.0028 by Student’s t test. (N) Quantification of 4-kDa FITC-dextran leakage in the in vitro ICH model in the presence of saline (control) or recombinant TIMP2 protein. n = 6 biological replicates. ***p = 0.0006 and ***p = 0.0007 at 30- and 60-min time points by Student’s t test, respectively. (O) Representative images of ZO-1 (green), claudin5 (green), and DAPI (blue) in primary HBMECs treated with saline (control) or recombinant TIMP2 protein at 48 h after in vitro ICH. Scale bars, 25 μm. (P) Representative images of caveolin-1 (green), meca32 (red), and DAPI (blue) in primary HBMECs treated with saline (control) or recombinant TIMP2 protein at 48 h after in vitro ICH. Scale bars, 25 μm. Data were shown as mean ± SD. EC, endothelial cell; FB, fibroblast; KD, knockdown. See also and .

Article Snippet: The following primary antibodies were used: mouse anti-claudin-5 (1:500, Invitrogen, 35–2500), rabbit anti-ZO-1 (1:500, Thermofisher, 61–7300), rabbit anti-caveolin-1 (1:1000, cell signaling, 3238S), rat anti-meca32 (1:200, Novus, NB100-77668), goat anti-TIMP2 (1:200, R&D, AF971), and mouse anti-actin (1:2000, Sigma, A5441).

Techniques: In Vitro, Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Control, Blocking Assay, Western Blot, Expressing, Transduction, shRNA, Sequencing, MANN-WHITNEY, Knockdown, Saline, Recombinant

Journal: Cell reports

Article Title: Fibroblasts repair blood-brain barrier damage and hemorrhagic brain injury via TIMP2

doi: 10.1016/j.celrep.2022.111709

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following primary antibodies were used: mouse anti-claudin-5 (1:500, Invitrogen, 35–2500), rabbit anti-ZO-1 (1:500, Thermofisher, 61–7300), rabbit anti-caveolin-1 (1:1000, cell signaling, 3238S), rat anti-meca32 (1:200, Novus, NB100-77668), goat anti-TIMP2 (1:200, R&D, AF971), and mouse anti-actin (1:2000, Sigma, A5441).

Techniques: Virus, Recombinant, Avidin-Biotin Assay, RNAscope, In Situ Hybridization, Software

Journal: PLoS ONE

Article Title: Lymphangiogenesis Is Required for Pancreatic Islet Inflammation and Diabetes

doi: 10.1371/journal.pone.0028023

Figure Lengend Snippet: MLDS treated BALB/c mice received FTY720, sunitinib, or anti-VEGFR3 mAb starting from the first STZ injection. (A) Whole mount immunohistochemistry of isolated normal islets of BALB/c mice. Blood vessels: CD31; lymphatic vessels: LYVE-1. Scale bar: 200 pixels. 200× magnification. (B) Immunofluorescent analysis of beta-cells (insulin), T cells (CD3) and lymphatic vessels (LYVE-1) in pancreas 7 days or 13 days after initiation of MLDS treatment. Scale bars: 32 µm. (D) Immunofluorescent analysis of beta-cells (insulin), blood vessels (MECA32) and lymphatic vessels (LYVE-1) 7 days after initiation of MLDS treatment. Scale bars: 32 µm. (C) and (E) Quantitative analysis of insulin, CD3, LYVE-1 and MECA32 staining of pancreas 7 days after initiation of MLDS treatment. 12–15 islets for insulin and CD3, 12–15 areas around islets for LYVE-1, and 14–21 islets or areas around islets for MECA32; 2 slides/mouse; 2–4 mice/group. * P≤0.05, ** P≤0.01, *** P≤0.001; ns , not significantly. (F) and (G) Normal BALB/c mice received indicated treatment for 7 days. (F) Immunofluorescent analysis of beta-cells (insulin), lymphatic vessels (LYVE-1) and blood vessels (MECA32). 200× magnification. (G) Quantitative analysis of LYVE-1 and MECA32 staining of pancreas. 18–21 areas around islets; 2 slides/mouse; 2–4 mice/group. P>0.1 vs untreated mice. Mean ± SD. 200× magnification.

Article Snippet: Purified rat anti-peripheral LN addressin (PNAd, MECA79), American hamster anti-CD3ε (145-2C11), rat anti-MECA32 and rat anti-CD31 (390) were from BD Biosciences-Pharmingen (San Jose, CA).

Techniques: Injection, Immunohistochemistry, Isolation, Staining