A22120 1

Rat brains were harvested following transcardial perfusion with normal saline and 4% paraformaldehyde under deep anesthesia 24‐h post‐HIBI. Specimens were fixed in 4% paraformaldehyde for 24–48 h, dehydrated in a graded ethanol series, and paraffin‐embedded. Coronal sections (3.5 μm thick) were then prepared. After deparaffinization and antigen retrieval, sections were probed with 10% fetal bovine serum in phosphate‐buffered saline (PBS) for 40 min and incubated with primary anti‐GPx4 (1:200; ab125066; Abcam) and anti‐NeuN (1:200; ab104224; Abcam) antibodies overnight at 4°C. Thereafter, sections were washed three times and incubated for 4 h at room temperature with tetramethyl rhodamine‐isothiocyanate‐conjugated goat anti‐mouse IgG (1:200; SA00007‐1; Proteintech) and fluorescein isothiocyanate‐conjugated goat anti‐rabbit IgG (1:200; A22120‐1; Abbkine, Wuhan, China) secondary antibodies. Nuclei were then stained with 4′,6‐diamidino‐2‐phenylindole (C1006; Beyotime Biotechnology) for 5 min, and immunofluorescence images were captured using a Nikon C1Si confocal microscope.

SSCs from the control and heat shock-treated groups were cultured in 24-well plates (0.8 × 10³ cells per well) and fixed with 4% paraformaldehyde at room temperature for 30 min. The cells were treated with 0.5% Triton X-100 at room temperature for 15 min and washed three times with PBS at room temperature for 5 min each time. Then, the cells were incubated with 10% goat serum at room temperature for 40 min. Then, the cells were incubated with polyclonal rabbit anti-GDNF receptorα-1 (GFRα1) (1:100; sc-10716, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or polyclonal mouse anti-Promyelocytic Leukaemia Zinc Finger (PLZF) (1:150, sc-22839, Santa Cruz Biotechnology) primary antibodies at 4 °C overnight. Finally, the cells were incubated with FITC- or TRITC-conjugated secondary antibodies (1:200, A22120-1, Abbkine, Wuhan, Chian) at 37 °C for 30 min and stained with DAPI for 5 min. We stained at least three sections for each group, and images were evaluated in at least three randomly selected fields per section under a magnification of × 400 with an inverted fluorescence microscope (IX53, Olympus, Tokyo, Japan).