*4.4. Histopathological Examination*

Harvested wounds together with the surrounding skins were used for the histological evaluation. The harvested samples collected on days 3, 7, and 14 were fixed in 10% buffered formalin solution for 24 h. The tissues were embedded in paraffin and sectioned into 5 μm thick slices for histopathological examination by hematoxylin and eosin (H&E) staining method. Then they were studied by a routine light microscope. The criterion that was studied in histopathological sections consisted of re-epithelialization, collagen deposition, fibroblast content, revascularizations, and inflammatory cells. Analysis of stained skin sections was performed by an experienced pathologist.

#### *4.5. Immuno-Histochemical Examinations*

The skin tissues including the wound site were excised and fixed in 10% buffered formalin for more than 24 h, then embedded in paraffin and cut into 5 μm thick slices. After deparaffinization and rehydration, antigen unmasking was performed as follows: the endogenous peroxidase of randomly selected section was inactivated by incubation with 3% hydrogen peroxide/methanol solution at 37 ◦C for 30 min. The slices were washed three times with PBS for 5 min each wash. In order to recover antigen, these sections were put into 10 mM citrate buffer solution (pH 6.0) and heated at 95 ◦C for 15 min, and then cool down at room temperature, followed by washing three times with PBS for 5 min each wash. The non-specific binding sites were blocked with 5% goat serum (Gibco, 16210072) for 10 min at 37 ◦C. After the redundant liquid was discarded, the sections were incubated with the following primary antibodies, respectively: anti-EGF antibody, anti-FGF antibody, and anti-CD31 antibody at 4 ◦C overnight and washed three times with PBS for 5 min, followed by incubation with biotinylated goat anti-rabbit secondary antibody kit (Santa Cruz, Shanghai, China) at 37 ◦C for 30 min, and then incubated with streptavidin-HRP for 30 min. The slides were dyeing with a DAB (3,3'-diaminobenzidine) solution, and then counterstained with hematoxylin and following by dehydration with sequential ethanol for sealing and microscope observation. FGF and EGF positive cells were analyzed from three identical areas in the dermal tissue per rat wound tissue section and analyzed for the statistical significance. Individual micro-vessels were counted at 200× magnification (0.152 mm2/field). For each section, three areas were selected from the vascularity of the wound tissues [38].
