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).