Anti nrf2 antibody 12721

Total protein was prepared using lysis buffer (0.1% triton X, TBS, phosphoblocker). After the separation of the cell lysate proteins by SDS‐PAGE electrophoresis, the cells were transferred to nitrocellulose membranes and blocked with 5% non‐fat milk. The membranes were then incubated overnight at 4°C with the following primary antibodies: Anti‐GPX4 antibody AB125066 (Abcam), anti‐NRF2 antibody #12721 (Cell Signaling Technology), and anti‐β‐actin (Sigma–Aldrich, St Louis). After incubation for 1 h at room temperature with peroxidase‐conjugated secondary antibody (Jackson Immuno Research Laboratories), the protein bands were examined using an ECL Western Blotting Detection Reagent and ImageQuant LAS 4000 mini software (GE Healthcare).

Sections of 4‐μm thickness were prepared using a microtome. The sections were deparaffinized, rehydrated, and subjected to antigen removal in 10 mM citrate buffer (pH 6.0; GPX4) or Tris‐EDTA buffer (pH 8.0; NRF2) at 95°C for 15 min. Endogenous peroxidase activity was blocked with 3% H₂O₂ in methanol for 5 min at room temperature. The sections were blocked with 10% goat serum and incubated with primary antibodies at 4°C overnight. Anti‐GPX4 antibody AB125066 (Abcam), anti‐NRF2 antibody #12721 (Cell Signaling Technology), anti‐4HNE antibody bs6313R (Bioss), and anti‐Ki67 ab16667 (Abcam) were diluted at 1:250, 1:100, and 1:600, respectively with 1% BSA/PBS. Then, the sections were incubated for 5 min with secondary antibody and peroxidase‐labeled streptavidin using SAB‐PO kits (Nichirei Biosciences). The sections were developed using DAB (Nichirei Biosciences). Finally, the sections were incubated with hematoxylin, dehydrated, and mounted. TUNEL‐HRP‐DAB staining (ab206386) was performed according to the manufacturer's instructions. Images were captured using a whole slide scanner (OliVIA VS120: Olympus Corp.) and quantified using TissueMorph software (VisioPharm).