CD31 Immunohistochemistry and Quantitation of Microvessel Density

Immunohistochemistry of tissue sections with rabbit anti-rat CD31 antibody (Santa Cruz Biotechnology, USA) was performed at 7 and 14 days to detect angiogenesis in the wound after intervention. Sections were deparaffinized, rehydrated, heated in a microwave oven twice for antigen recovery, treated with 3% H₂O₂ and then incubated with 5% goat serum albumin. Then, the sections were incubated overnight at 4°C with primary rabbit anti-rat CD31 antibody (1:75, Santa Cruz Biotechnology), followed by a 1-h incubation with HRP-conjugated goat anti-rabbit secondary antibody (1:200, Abcam) and visualization with a DAB kit (ZSGB-BIO, China). After being counterstained with hematoxylin, the slides were assessed with a fluorescence microscope (40FL Axioskop, Zeiss).

The three regions with the most neovascularization at low magnification (5X) on each slide were selected. Then, three randomly selected areas in each region were imaged at 20X magnification. These images were analyzed with Image-Pro Plus software. The mean number of blood vessels per image was defined as the microvascular density (MVD).