Caspase 8

Deparaffinized and rehydrated liver tissue sections were immersed in citrate buffer for antigen retrieval and then incubated with 3% hydrogen peroxide for 10 min to quench endogenous peroxidase activity. Next, sections were blocked with 10% goat serum albumin and incubated with a primary antibody targeting alpha-smooth muscle actin (α-SMA, 1:100; Abways, Shanghai, China), interleukin-1 beta (IL-1β, 1:100; ABclonal, Wuhan, China), YAP (1:100; Santa Cruz Biotechnology, Inc., California, USA), P-YAP (1:100; Abways, Shanghai, China), glutathione peroxidase 4（GPX4, 1:100; Abways, Shanghai, China), solute carrier family 7 members 11 (SLC7A11, 1:250; Proteintech, Wuhan, China), receptor-interacting serine-threonine kinase 3 (RIPK3, 1:100; Signalway antibody, College Park, USA), or caspase8 (1:200; Abcam, Cambridge, UK) at 4 °C overnight. After washing with phosphate-buffered saline (PBS) three times, sections were incubated with the corresponding horse radish peroxidase (HRP)-conjugated secondary antibody at room temperature for 30 min. After washing, sections were visualized with 3,3′-diaminobenzidine (DAB) and stained with hematoxylin. Images were captured and integral optical density (IOD) was determined using Image-Pro Plus 6.0 software. A total of ten non-overlapping fields were analyzed from each specimen.

Kidney tissues were lysed in RIPA buffer, run on a 10% SDS-polyacrylamide electrophoresis gel and transferred onto a nitrocellulose membrane (Hybond C Extra, Amershan Biosciences, Little Chalfon, USA). The membrane was incubated in a blocking buffer A (PBS, 5% nonfat milk and 0.1% Tween-20) and incubated overnight at 4 °C with primary rabbit anti-rat Bax (diluted 1:300; Abcam, USA), Bcl-2 (diluted 1:300; Abcam, USA), Caspase-3 (diluted 1:200; Abcam, USA), Caspase-8 (diluted 1:300; Abcam, USA) and Caspase-9 (diluted 1:200; Abcam, USA) antibody. Then the membrane was washed once for 15 min and twice for five min in PBS, followed by a peroxidase-conjugated sheep anti-rabbit IgG (Santa Cruz Biotechnology) at a 1:10000 dilution. At last, the membrane was developed with a chemiluminescent agent (ECL). Each membrane was stripped and probed with mouse primary anti-β-actin antibody (Sigma, USA) to confirm and estimate the loading and the transfer. We used a bio-image analysis system (Bio-Rad, USA) to analyze the bands.