Collagen 1

Hematoxylin and eosin (H&E) staining, Masson’s trichrome staining, immunohistochemistry, and immunofluorescence were performed as described previously [18 (link)]. For immunohistochemical staining, the sections were incubated with primary antibodies against GPX4 (1:100; Affinity Biosciences, China), collagen I (1:100; Servicebio, China), TGF-β1 (1:100; Servicebio, China), CTGF (1:100; Servicebio, China), and PDGF (1:100; Servicebio, China) overnight at 4 °C. The sections were then washed and incubated at room temperature with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:100; Sigma-Aldrich, USA). For immunofluorescence staining, slide-mounted tissues were blocked with 5% bovine serum albumin for 30 min and then incubated overnight at 4 °C with antibodies specific for E-cadherin (1:100; Sigma-Aldrich, USA), GPX4 (1:100; Affinity Biosciences, China), and alpha-smooth muscle actin (α-SMA, 1:100; Abcam, UK), followed by staining with corresponding secondary antibodies (1:200, Beyotime, China) at room temperature. Cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; Proteintech, China). Images were acquired using a microscope with a mounted camera (Nikon, Tokyo, Japan).

Mice lung tissue was fixed, embedded in paraffin, sectioned to 5-μm-thick slices, deparaffinized, and dehydrated. Endogenous peroxidase activity was quenched in 0.3% H₂O₂ for 10 min, and the slides were blocked with 5% bovine serum albumin (BSA) for 30 min. The sections were stained with primary antibodies against α-SMA (1:100; Servicebio, Wuhan, China), collagen I (1:100; Servicebio, Wuhan, China) or collagen III (1:100; Servicebio, Wuhan, China) overnight at 4 °C, followed by incubation with an HRP-conjugated secondary antibody (1:100; Sigma-Aldrich, MI, USA) at room temperature (20–25 °C) for 30 min. Images were acquired by a microscope (Nikon, Tokyo, Japan).