Af5103

To demonstrate the proliferation of epidermal cells and detect the inflammatory response, immunohistochemistry stain was performed according to a previous study [26 (link)]. The sliced tissues were incubated with primary antibodies, including rabbit anti-mouse interleukin-1β (IL-1β, Affinity; AF5103, China; 1:100), interleukin-6 (IL-6, Servicebio; GB11117, China; 1:600), tumor necrosis factor α (TNF-α, Servicebio; GB11188, China; 1:500), and Ki67 (Servicebio; GB11499, China; 1:500) at 4 °C for 12 h. Secondary antibodies (Servicebio; GB23303, China; 1:400) were used for incubation at 37 °C for 1 h, and diaminobenzidine staining (Biosharp, China) was performed. The positive staining of IL-1β, IL-6, TNF-α, and Ki67 was recorded by light microscopy (Primo Star, Zeiss, Germany) and measured by ImageJ software.

Immunohistochemical staining was performed to investigate epidermal cell proliferation and inflammatory factors expression in regenerating skin tissue, as per previous study [46 (link)]. Primary antibodies, including rabbit anti-mouse IL-1β (Affinity, af5103, China, 1:100), IL-6 (Servicebio, gb11117, China, 1:600), TNF-α (Servicebio, gb11188, China, 1:500), and Ki67 (Servicebio, gb11499, China, 1:500) were used following the provided instructions. After incubation, the skin wound tissue was incubated with secondary antibodies (Servicebio, GB23303, China, 1:400) for 1 h at 37 °C, then sealed and observed by light microscopy (Primo Star, Zeiss, Germany). ImageJ software was used to measure positive staining intensity and analyzed by GraphPad Prism software.