Anti gpx4

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.

BMSCs were fixed using a 4% paraformaldehyde solution (4 °C, 25 min), permeabilized with 0.5% Triton X-100 (room temperature, 20 min), blocked with 5% sheep serum (37 °C, 20 min), and washed with PBS at each step, except for the final step. They were then incubated with specific antibodies: anti-ALP (1:200, HUABio, Zhejiang, China), anti-RUNX2 (1:200, HUABio, Zhejiang, China), anti-OSX (1:200, HUABio, Zhejiang, China), anti-GPX4 (1:200, HUABio, Zhejiang, China), anti-ACSL4 (1:200, HUABio, Zhejiang, China), and anti-NRF2 (1:200, HUABio, Zhejiang, China) overnight at 4 °C. The next day, BMSCs were incubated with a secondary anti-rabbit antibody (1:200, Invitrogen, Carlsbad, USA) at 37 °C for 1 h. The cytoskeleton was stained with phalloidin (37 °C, 20 min), and the Nucleus was stained with DAPI (37 °C, 10 min), followed by cleaning with PBS at each step. Images were captured using a confocal microscope (Olympus, Tokyo, Japan).