The mice were euthanized 30 days after exposure to IR. The skin was fixed in a formaldehyde solution (# 50-00-0; Damao Chemical Reagent Factory, Tianjin, China) and prepared for histological analysis. Samples were dehydrated, embedded in paraffin, cut into 5 μm micrometer slices using a microtome and mounted on glass slides. Samples were stained with hematoxylin and eosin (H&E; # G1080 and # G1100; Solarbio, Beijing, China) and Masson’s Trichrome (# G1345; Solarbio). Images were captured using a Zeiss Axio Lab.A1 upright microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany).
To detect the tissue proliferation marker Ki67, sections were also immunostained. Briefly, sections were incubated with rabbit anti-Ki67 primary antibody (ab15580; Abcam, Cambridge, UK). Slides were subsequently stained with biotinylated goat anti-rabbit secondary antibody (# PV-9000; ZSGB-BIO, Beijing, China). Reaction sites were visualized with DAB (ab64238; Abcam), and the slides were counterstained with hematoxylin. The number of Ki67-positive cells was assessed by random selection of a field of view per sample using a Zeiss Axio Lab.A1 upright microscope.
To detect the tissue proliferation marker Ki67, sections were also immunostained. Briefly, sections were incubated with rabbit anti-Ki67 primary antibody (ab15580; Abcam, Cambridge, UK). Slides were subsequently stained with biotinylated goat anti-rabbit secondary antibody (# PV-9000; ZSGB-BIO, Beijing, China). Reaction sites were visualized with DAB (ab64238; Abcam), and the slides were counterstained with hematoxylin. The number of Ki67-positive cells was assessed by random selection of a field of view per sample using a Zeiss Axio Lab.A1 upright microscope.