Rat anti cd31 antibody

Heart sections were deparafinized, rehydrated, and subjected to antigen retrieval in pH 6.0 citrate buffer in a pressure cooker for 10 min. Sections were then permeabilized in 0.1% Triton/PBS for fifteen minutes. Sections were blocked with 10% goat serum for 30 min followed by incubation with rat anti‐CD31 antibody (1:20 dilution, Dianova, Hamburg, Germany) overnight at 4°C. Cy3 conjugated anti‐rat IgG (1:500 dilution, Sigma‐Aldrich) was then applied for 1 h at room temperature. To decrease autofluorescence of the cardiac tissue, sections were treated with 0.1% Sudan Black (Sigma‐Aldrich) dissolved in 70% ethanol for 30 min. Sections were counterstained with DAPI (Vector Laboratories) and imaged with a Zeiss ApoTome microscope. At least 16 images (20X) in the peri‐infarct region were obtained from each heart (from six sections). NIH Image J software was utilized to determine the number of capillaries, average capillary size, and number of nuclei per field.

Kidneys were immersed in 10% formalin and embedded in paraffin. Sections (4 μm thick) were stained with hematoxylin/eosin or Masson's trichrome and processed for histopathology or immunohistochemistry. For immunohistochemistry, paraffin-embedded sections were stained with rabbit anti-nytrotyrosine primary antibody (Upstate, number 06284, 1 : 200), rabbit anti-CD3 antibody (DAKO, number A0452), and rat anti-F4/80 antibody (AbD Serotec, number MCA497R) then the staining was revealed with Histofine reagent (Nichirei Biosciences) and slides were counterstained with hematoxylin. For immunofluorescence, paraffin-embedded sections were stained with a guinea pig anti-nephrin antibody (Progen) and a rat anti-CD31 antibody (Dianova, number SZ31) then the stainings were revealed with a goat anti-guinea pig alexa568-coupled antibody and a donkey anti-rat alexa488-coupled antibody (Invitrogen) and nuclei were counterstained with DAPI. Photomicrographs were taken with an Axiophot Zeiss photomicroscope. Staining surface quantifications were performed with a macro designed on ImageJ.

Paraffin sections were stained with hematoxylin and eosin (HE) and Masson’s trichrome (MT). For immunohistochemical analysis, rabbit anti-α-smooth muscle actin antibody (αSMA: Abcam plc, Tokyo, Japan), rabbit anti-macrophage antibody (Mac-1: CD11b, Abcam plc), rat anti-neutrophil antibody (NINP-R14: Hycult Biotech, Uden, Netherlands), rat anti-CD31 antibody (Dianova GmbH, Hamburg, Germany), and rat anti-PI3 kinase p110 beta antibody (PI3K: Abcam plc) were used. Heat-antigen-activations in 0.01 M citrate buffer (pH 6.0) for CD31 antibody and Tris/EDTA buffer (pH9) for PI3K antibody were performed. For rabbit antibodies, Envison kit (Dako Japan Inc., Tokyo, Japan), and for rat antibodies, N-Histofine Simple Stain Mouse MAX PO (rat) detection system (Nichirei Bioscience Inc., Tokyo, Japan), were used. Colorization was performed with 3,3-diaminobenzidine. Hematoxylin was also used for a nucleus counter stain.