Sa00007 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.

The hippocampal tissues of the mice from different groups were treated with normal saline, fixed with 4% paraformaldehyde for 24 h, treated with 0.1 M PBS and subsequently dehydrated in graded ethanol after being embedded in paraffin. An immunofluorescence assay was then performed on 3.5 μm-thick coronal sections. Following retrieval, the sections were incubated in 0.1 M PBS containing 10% serum for 40 min and incubated overnight at 4 °C with primary antibodies: mouse monoclonal antibody [Mec-168] against MeCP2 (ab50005, 1:1000, Abcam, Inc., Cambridge, UK) and rabbit polyclonal antibodies against SPRY2 (ab85670, 1:1000, Abcam, Inc., Cambridge, UK) and NeuN (mouse 1:1,000; 2,742,283, Millipore, Billerica, MA, USA). After three 0.1 M PBS washes (5 min per wash), the sections were incubated with fluorescein isothiocyanate (FITC)-conjugated secondary goat anti-rabbit immunoglobulin G (IgG) (1:100; SA00003-2, Proteintech Group, Chicago, IL, USA) or tetramethylrhodamine isothiocyanate-conjugated anti-mouse IgG (1:100; SA00007-1, Proteintech Group, Chicago, IL, USA) at room temperature for 4 h. The sections were counterstained with 4’,6-diamidino-2-phenylindole for 5 min. Immunofluorescence images were obtained using a Nikon Eclipse NI microscope.