Before the histological procedure, the slides were incubated in xylene and then hydrated in 100% ethanol and 95% ethanol for removal of paraffin. Slides were stained with Harris hematoxylin and eosin for inflammatory cells, and Masson Trichrome for collagen fiber.
For immunohistochemical staining of FGF2, the slides were incubated in 3% hydrogen peroxide for 10 minutes, washed in wash buffer (TBS-T, Tris Buffered Saline containing 0.1% Tween-20), and incubated in blocking solution (TBS-T with 5% normal goat serum) for 1 hour. Then, the rabbit polyclonal FGF2 antibody (Santa Cruz, CA, USA) was diluted in blocking solution and added to each section. These solutions were incubated overnight at 4℃. The sections were then rinsed with wash buffer and incubated with biotinylated anti-rabbit IgG (Vector Laboratories Inc., Burlingame, CA, USA) for 1 hour. Slides were incubated with ABC reagent (Vector Laboratories Inc., Burlingame, CA, USA) and incubated in 0.02% diaminobenzidine and 0.003% hydrogen peroxide in 1M Tris-buffered saline (pH 7.5). As soon as the sections were developed, the slides were immersed in distilled water. The sections were counterstained with hematoxylin. After staining, slides were dehydrated in 95% and 100% ethanol, incubated in xylene, and mounted. Pictures of the skin layers were taken using a bright-field microscope (BX51; Olympus, Tokyo, Japan).
For immunohistochemical staining of FGF2, the slides were incubated in 3% hydrogen peroxide for 10 minutes, washed in wash buffer (TBS-T, Tris Buffered Saline containing 0.1% Tween-20), and incubated in blocking solution (TBS-T with 5% normal goat serum) for 1 hour. Then, the rabbit polyclonal FGF2 antibody (Santa Cruz, CA, USA) was diluted in blocking solution and added to each section. These solutions were incubated overnight at 4℃. The sections were then rinsed with wash buffer and incubated with biotinylated anti-rabbit IgG (Vector Laboratories Inc., Burlingame, CA, USA) for 1 hour. Slides were incubated with ABC reagent (Vector Laboratories Inc., Burlingame, CA, USA) and incubated in 0.02% diaminobenzidine and 0.003% hydrogen peroxide in 1M Tris-buffered saline (pH 7.5). As soon as the sections were developed, the slides were immersed in distilled water. The sections were counterstained with hematoxylin. After staining, slides were dehydrated in 95% and 100% ethanol, incubated in xylene, and mounted. Pictures of the skin layers were taken using a bright-field microscope (BX51; Olympus, Tokyo, Japan).