Pr30012

Paraffin‐embedded lung cancer puncture specimens were sectioned into 4 µm sections for immunohistochemical staining. Haematoxylin and eosin staining was used for initial tissue sectioning. Subsequently, morphologically representative tumour areas were re‐sectioned. The sections were dewaxed with xylene and ethanol, followed by inhibition of endogenous peroxidase with a 3% hydrogen peroxide solution and incubation with 1% BSA at room temperature for 10 min. Incubation at 4°C overnight was carried out with primary antibodies: Anti‐SPP1 monoclonal antibody (1:100; LOT: #39o4953; affinity biosciences), Anti‐AGL (1:100; LOT: BB12027175; affinity biosciences), Anti‐CD44 (1:100; LOT: #37o8571; affinity biosciences), Anti‐cMET (1:100; LOT: #39o4953; affinity biosciences), Anti‐HGF (1:100; LOT: #44o6892; affinity biosciences).
Subsequently, secondary antibody treatment (Horseradish peroxidase anti‐mouse; PR30012, Proteintech) was conducted for 20 min at room temperature. Specific signals were visualised using diaminobenzidine substrate (DAB). Positive immunohistochemical manifestations, characterised by intracellular target proteins stained with brownish‐yellow granules, were counted in one randomly selected high magnification field of view, and the positive rate was calculated. On average, tens of fields of view were counted in each group (Supplementary Data 2).

We collected thymus for protein extraction using RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with protease inhibitors (Beyotime, Shanghai, China). The protein from the thymus was separated by electrophoresis. Transfer the protein to the PVDF membrane at 4 °C. After blocking with 5% skimmed milk at room temperature for 2 h, the membranes were incubated with GPX4 primary antibody (ab40993, Abcam, Waltham, MA, USA) and GAPDH primary antibody (60004-1-Ig, Proteintech, Wuhan, China) at 4 °C overnight. After incubation with peroxidase-conjugated secondary antibodies (PR30011 and PR30012, Proteintech, Wuhan, China) for 1 h at room temperature. The signal was detected on an ImageQuant LAS 500 (GE, Chicago, IL, USA) using the ECL Kit (Biosharp, Hefei, China). GAPDH expression served as a loading control for quantification.