Harris hematoxylin

For HE staining, 5-µm transverse sections (8 µm) of tissue at 7 and 14 days post surgery in each group were deparaffinized and put into fresh xylene for 15 min twice. Sections were rehydrated in 100% alcohol for 5 min twice, then 95% alcohol and 70% alcohol once for 3 min. Subsequently, sections were washed briefly in ddH₂O, stained in Harris hematoxylin (Beyotime Institute of Biotechnology) solution for 5 min. Sections were then washed in running tap water for 8 min and differentiated in 1% acid alcohol for 30 sec, blued in 0.2% ammonia water for 30 sec. The sections were washed in running tap water for 5 min and rinsed in 95% alcohol ~15 times. Sections were stained in Eosin-Phloxine (Beyotime Institute of Biotechnology) solution for 1 min then dehydrated through 95 and 100% alcohol (5 min each) and cleared in two changes of xylene (5 min each). Finally, the sections were mounted with mounting medium (Beyotime Institute of Biotechnology). Images were captured using a FV 300 confocal microscope (Olympus Corporation, Tokyo, Japan).

Mouse liver tissues were fixed in 4% paraformaldehyde solution at 37°C for 24 h and embedded in paraffin, then sectioned at 5 µm thickness. The sections were stained with Harris hematoxylin (Beyotime Institute of Biotechnology) at 37°C for 5 min and eosin (95% ethanol preparation, Beyotime Institute of Biotechnology) at 37°C for 15 sec. After staining, the liver tissues were observed under a light microscope at ×200 magnification. For the immunofluorescence detection of liver tissues, paraffin sections were dewaxed and hydrated, and immersed in citrate buffer for antigen retrieval. Sections were blocked with 5% BSA at 37°C for 30 min, and incubated with GPX4 (1:200) and HMGB1 (1:200) antibodies at 4°C overnight. Slices were then incubated with the Cy3 and FITC-labeled secondary antibody at 37°C for 1 h in a dark room. DAPI (5 µg/ml) was used to stain nuclei at 37°C for 5 min in dark room. Finally, the slices were imaged under an inverted fluorescence microscope at ×200 magnification.