Anti p21

Mouse lung tissue was fixed in 4% paraformaldehyde for 24 h, followed by gradient dehydration, paraffin embedding, sliced, and antigen retrieval. The tissue sections were then blocked with BSA (G5001, Servicebio) and incubated with primary antibodies anti-P21 (1:400, GB11153, Servicebio), anti-pro-SPC (1:2000, ab211326, Abcam, Cambridge, UK), and anti-F4/80 (1:5000, Abcam), and counterstained with hematoxylin. The images were captured using a Leica DFC7000T microscope and the positive area was analyzed using ImageJ software.

The paraffin blocks containing the skin samples were sectioned into 5 μm slices and heat-treated for 2 h at 65 °C. A citrate buffer (pH 6.0) was used for antigen retrieval at 121 °C for 2 min. After a blocking process with serum and hydrogen peroxide, the slices were incubated overnight at 4 °C with the anti-p21 (1:1000, Servicebio, Wuhan, China) and anti-p53 (1:200, Bioss, Beijing, China) primary antibodies. Next, the slices were incubated with a horseradish enzyme-labeled goat anti-rabbit immunoglobulin G antibody (1:200, Servicebio, Wuhan, China) at 37 °C for 30 min, stained with hematoxylin and 3,3′-diaminobenzidine. The stained slices were examined using an optical microscope (NIKON ECLIPSE E100, Nikon, Tokyo, Japan), and images were captured using a camera (NIKON DS-U3, Nikon, Tokyo, Japan). Finally, Image J software version 1.53 was employed to determine the positive area.