Anti gpx4 antibody

The testes were fixed in 4% FPA overnight and sectioned into slices of 2–3 μm. The sections were rinsed three times in PBS, and endogenous peroxidase was blocked using 3% H₂O₂ at RT for 15 min. Thereafter, sections were blocked using 5% BSA in 0.3% Triton X-100 in PBS for 1 h at RT. Subsequently, we inoculated the sections overnight with anti-GPX4 antibody (Proteintech, Wuhan, China, 14432-1-AP, 1:1,000 dilution) at 4 °C. Then, we rinsed the sections three times in PBS and inoculated them with a secondary antibody for 1 h at RT. Positive immunoreactivity was performed using DAB and assessed by microscopy (Olympus, Tokyo, Japan). Images were analyzed using Image-Pro Plus 6.0 (Media Cybernetics, MD, US).
For fluorescent staining, the sections were blocked with 3% BSA for 30 min and incubated overnight with anti-GPX4 antibody (14432-1-AP, 1:100, Proteintech Group, Inc) and anti-FPN1 antibody (Cincinnati, OH, US, DF13561) at 4 °C. The following morning, the sections were rinsed three times in PBS and incubated with fluorescent secondary antibodies (SA00013-3, Proteintech, Wuhan, China) for 1 h in the dark at RT. Subsequently, the sections were rinsed in PBS, counterstained with DAPI (Wellbio, Guangzhou, China) and mounted using Prolong Gold anti-fade (Servicebio, Wuhan, China). The samples were then evaluated using a microscope (Olympus, Tokyo, Japan).

Whole-cell extraction and western blotting were performed as previously described.^{17 (link)} Anti-GPX4 antibody was purchased from Proteintech (Manchester, UK). Anti-PARP, caspase 8, caspase 3, cleaved caspase 3, MLKL, pMLKL, p53 and SLC7A11 antibodies were sourced from Cell Signaling Technology (Danvers, MA, USA).