Immunohistochemistry

Lung tissues of mice at 24 h following IR were fixed in 4% paraformaldehyde fixative solution, and then sliced at thickness of 5 µm after embedding in paraffin. Paraffin sections of lung tissues were deparaffinized and antigen retrieval was then undertaken using Tris-EDTA Buffer (pH 9.0) at 100°C for 30 min. Endogenous peroxidase was then blocked using 0.3% hydrogen peroxide methanol solution. Sections were incubated overnight with rabbit primary antibodies, which included anti-PIEZO1 (Abcam, ab128245, 1:50), anti-GPX4 (Abcam, ab125066, 1:100), anti-VE-cadherin (Cell Signaling Technology, #2500,1:50), and anti-SCL7A11 (Abcam, ab37185, 1:100). Horseradish peroxidase-conjugated goat anti-rabbit IgG was used as the secondary antibody. Sections were dehydrated and fixed after counterstaining with hematoxylin. Sections were chemically developed based on the DAB method, and then observed and photographed under microscope.