2.11 Immunohistochemical analysis of wound cell proliferation
At 3 and 14 d, wound tissue specimens harvested from wound-healing model rats were used for immunohistochemical evaluation. Immunohistochemical staining of vascular endothelial growth factor (VEGF) and CD31 were performed using a streptavidin-biotin method. In brief, sections were dewaxed and microwaved for 10 min to retrieve antigens, and then endogenous peroxidase was blocked by incubation in 3% hydrogen peroxide for 30 min in the dark. Sections were permeabilized with 1% Triton solution and blocked in 5% bovine serum albumin. Next, sections were incubated with antibodies against VEGF (1:300 dilution, GB14165, Google Biotechnology) and CD31 (1:200 dilution, GB11063-3, Google Biotechnology) overnight. After rinsing with PBS, sections were incubated with rabbit secondary antibody for 40 min. After washing four times with PBS, DAB and hematoxylin were applied for coloration and re-dying of the nucleus, respectively. Finally, sections were dehydrated and sealed with PermountTM Mounting Medium for microscopic observation (Olympus IX71, Tokyo, Japan).