Rabbit anti rat cd31 antibody

Histological analysis and immunohistochemistry staining were performed as previously described [38 (link)]. OCT-embedded common carotid arteries were cut systematically in serial 5-μm cross sections using a cryotome (Leica CM3050S, Leica Microsystems, Germany). Analysis was carried out in the injured left common carotid artery, whereas the contralateral served as a control. For morphometric analysis, sections were stained with hematoxylin and eosin (HE). For immunofluorescence analysis, sections were stained with a rabbit anti-rat CD31 antibody (1:250, Abcam, USA) and visualized using an Alexa Fluor 647 mouse-anti-rabbit IgG secondary antibody (1:500, eBioscience). For immunohistochemistry analysis, sections were stained with a rabbit anti-rat CD31 antibody (1:150, Abcam, USA) and visualized using a mouse-anti-rabbit secondary antibody (1:500, Sigma). Negative controls using IgG controls matching in species and concentration were run in parallel. Pictures were taken with a microscope (Leica CM3050S, Leica Microsystems, Germany) and a digital camera (DFC 320, Leica Microsystems) at ×100 magnification. Morphometric analysis was performed per sample followed by computer-assisted morphometric analysis (ImageJ, NIH, USA). Subsequent morphometric analyses were performed in a double-blinded manner. Four animals per group and 3 samples per animal were analyzed.

The expression of CD31 known as platelet-endothelial cell adhesion molecule-1 was detected by immunofluorescence staining in each group. Briefly, the paraffin sections were placed in an oven at 60 °C for 1 hour and then rehydrated in a graded ethanol series (100 %, 95 %, 90 %, 85 %, and 70 %). Citrate buffer was used for heat-induced antigen retrieval. Then goat serum was used as a blocking solution before the primary antibody was applied. The sections were incubated overnight in 4 °C with rabbit anti-rat CD31 antibody at a 1:150 dilution (Abcam, USA), and mouse anti-rat Ki-67 (a marker of proliferation) antibody at a 1:100 dilution (Abcam, USA).The secondary antibody, goat anti-rabbit antibody conjugated to Alexa Fluor 594 (BA1032, China) and goat anti-mouse antibody conjugated to Alexa Fluor 488 (Life, USA) were applied at 1:200 dilutions for 1 hour. Then immunofluorescence photomicrographs were obtained at 400× magnifications using a fluorescence microscope (Leica, Solms, Germany), and the intensity of the CD31-positive signal was quantified with Image pro plus 6.0 image analysis software (Media Cybernetics, USA) to represent glomerular endothelial cell proliferation.