Antifade

After dewaxing and rehydration, paraffin sections were treated with Citrate Antigen Retrieval Solution (BBI E673001), and then blocked with blocking buffer (2% FBS, 3% BSA, and 0.2% Triton X‐100 in PBS). The sections were incubated with primary antibodies diluted in blocking buffer overnight at 4°C (isotype‐matched antibodies were used as controls). After washing thrice with PBST (PBS with 0.2% Triton X‐100), the sections were incubated with secondary antibodies diluted with blocking buffer for 2 h at 25–30°C in the dark. The nuclei were stained with a DAPI staining solution (Servicebio G1012) for 10 min. Slides were mounted with Antifade (Servicebio G1401). The IF‐stained sections were examined using digital slide scanning (Pannoramic DESK, P‐MIDI, P25) and analyzed using CaseViewer 2.4 (3DHISTECH).

The paraffin sections of mice brains were prepared as above. After antigen retrieval, the tissue sections were blocked with blocking buffer, then incubated with the primary antibody Anti-GPX4 (ET1706-45, HUABIO, China) and Anti-NeuN (66836-1-Ig, Proteintech, China), or Anti-CD8 (GB13429, Servicebio, China) and Anti-CD3 (60181-1-Ig, Proteintech, China) overnight at 4 °C, and with the secondary antibody for 1 h at room temperature, including Cy3 conjugated Goat Anti-Rabbit IgG (GB21303, Servicebio, China) and FITC conjugated Goat Anti-Moue IgG (GB22301, Servicebio, China). The nuclei were stained with DAPI staining solution (G1021, Servicebio, China) for 10 min. Finally, the slides were mounted in Antifade (G1401, Servicebio, China) and examined using digital slide scanning (Pannoramic DESK, P-MIDI, P25), and analyzed using CaseViewer 2.4 (3DHISTECH). The relative expression of GPX4 and NeuN was calculated using fluorescent density, analyzed through ImageJ software, and then standardized to DAPI count.