Rabbit anti human fibrin fibrinogen antibody

For immunohistochemistry, rabbit anti-human cleaved caspase 3 antibody (1:300, Cell Signaling Technology, Danvers, MA), rabbit anti-human hypoxia-inducible factor 1a antibody (1:1000, Novus Biologicals, Littleton, CO), anti-human 4-hydroxy-2-nonenal antibody (10 µg/ml, Japan Institute for the Control of Aging, Japan), rabbit anti-human HO-1 antibody (1:500, Enzo Life Sciences, Farmingdale, NY), rabbit anti-human fibrin/fibrinogen antibody (1:4000, Dako, Denmark), and rat anti-mouse MOMA2 antibody (1:400, AbD Serotec, Raleigh, NC) were used. TUNEL stain kit was from Wako chemicals (Osaka, Japan). Neutrophils were visualized using Naphthol AS-D chloroacetate Esterase stain (Muto Pure Chemicals, Tokyo, Japan). About 5 consecutive fields were examined in each slide at 100 or 200-fold magnification. All assessments were performed with ImageJ (National Institutes of Health, Bethesda, MD).

For immunohistochemistry, rat anti-mouse CD31 antibody (0.3125 μg/ml, BD Pharmingen, Franklin Lakes, NJ), goat anti-mouse VEGF antibody (0.33 μg/ml, R&D Systems, Inc., Minneapolis, MN), goat anti-human nephrin antibody (0.4 μg/ml, Santa Cruz Biotechnology, Dallas, TX), goat anti-mouse tissue factor (4 μg/ml, R&D Systems, Inc., Minneapolis, MN), rabbit anti-human fibrin/fibrinogen antibody (1.8 μg/ml, Dako, Denmark), and rat anti-human/mouse galectin3 (MAC2) antibody (0.5 μg/ml, ebioscience, San Diego, CA) were used. Heat–induced antigen retrieval was performed using sodium citrate buffer to detect VEGF, fibrin, and MAC2. Proteinase K (Dako, Denmark) was used to detect CD31, nephrin, and tissue factor. Primary antibodies were incubated overnight at 4 °C. N-Histofine simple stain kits (Nichirei biosciences Inc., Tokyo, Japan) were used as a secondary antibody according to the manufacturer’s protocol. We incubated sections with IgG isotype or without primary antibody as a negative control. Glomerular density of each protein was assessed using Image J (National Institute of Health, Bethesda, MD).