Anti ripk1

Formalin-fixed, paraffin-embedded sections were cut into a thickness of 4 μm and mounted on Matsunami adhesive silane-coated glass slides (Matsunami Glass, Osaka, Japan). After deparaffinization and rehydration, the slides were autoclaved in 10 mM citrate buffer for 20 min to retrieve the antigens. Then, endogenous peroxidase was quenched with 0.3% hydrogen peroxide (H₂O₂) in methanol at room temperature for 10 min. After blocking, the sections were incubated at 4 °C overnight with the following primary diluted antibodies: anti-CYP2E1 (dilution, 1:500; Abcam, Cambridge, UK), anti-RIPK1 (dilution, 1:200; Santa Cruz Biotechnology, Dallas, TX, USA), and anti-RIPK3 (dilution, 1:300; Imgenex, San Diego, CA, USA). Subsequently, the sections were incubated with peroxidase-labeled polymer conjugated secondary antibody (Dako Japan, Tokyo, Japan) for 30 min at room temperature. Immunoreactivity was detected with a diaminobenzidine substrate kit (Dako Japan), and the sections were counterstained with hematoxylin. ImageJ imaging analysis software (National Institutes of Health, Bethesda, MD, USA) was used to quantitate the percentage of necrotic area. Field images at ×100 magnification were selected at random from different individuals. The percentage of necrosis was determined by measuring the total dimension of the field and comparing it with the dimension of the necrotic area.