Photodocumentation system

Twenty μg from each sample were diluted in Laemmli loading buffer, denatured by boiling for 5 minutes, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Biorad, Hercules, CA). Membranes were blocked for 2 hrs in TBS containing 0.1% Tween 20 (Sigma-Aldrich) and 5% nonfat dry milk (Bio-Rad). After an overnight incubation at 4°C with the primary antibodies in TBS, 0.1% Tween 20, and 5% nonfat dry milk or bovine serum albumine (BSA), blots were washed three times with TBS containing 0.1% Tween (TBS-T) and incubated with a HRP-linked secondary antibodies for one hour. After three washes with TBS-T, the membranes were treated with ECL detection system (GE Healthcare, Piscataway, NJ), exposed to x-ray film (Denville Scientific, Metuchen, NJ) and developed to visualize the labeled protein bands. Molecular mass was estimated by comparison of sample bands with prestained molecular mass marker (Bio-Rad). For quantitative studies, the bands on x-ray films were scanned using a photodocumentation system (Alpha Innotech) and analyzed with ImageQuant 5.2 software (GE Healthcare). Where indicated, blots were stripped and reblotted with the corresponding antibody. Values were normalized respect to b-actin or total antibody as indicated.

The skin tissues around the wound were harvested on the 8th day after the injury and then dissolved by using the RIPA buffer (Beyotime, China). The supernatant was subsequently harvested through centrifugation at 12,000 rpm, at 4°C for 15 min, and the concentration of protein was determined via the BCA Protein Assay Kit (Solarbio, China). Each channel is loaded with an equal amount of protein lysate isolated on an acrylamide gel (10%) and subsequently transferred to the membrane of PVDF. After blocking, corresponding primary antibodies, including VEGF, bFGF, and GAPDH, were incubated at 4°C for 12 h. The GAPDH expression was exploited as a control. Images were captured by Alpha Innotech Photo-documentation System.