Peroxidase conjugated sheep anti rabbit igg

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.