Peroxidase labeled goat anti rabbit igg

The homogenate was made from the brain tissue obtained after reperfusion, which was further centrifuged (13200×g, 20 min, 4°C). The protein contained in supernatant was evaluated by BCA protein assay kit, standardized by bovine serum albumin (KeyGEN, Nanjing, China). The nitrocellulose membrane (Hybond ECL, Amersham Pharmacia Biotech, U.S.A.) was exerted to receive the samples (50 μg each) separately, followed by blockage of nonfat milk for 2 h before the respective overnight incubation with anti-phospho-p53 (Ser¹⁵) antibody (Cell Signaling Technology, Woburn, MA, U.S.A.) (1/1000 diluted), PUMA (Cell Signaling Technology),(1/1000 diluted), DRAM (Assay Designs and Stressgen Bioreagents, Ann Arbor, MI, U.S.A.) (1/1000 diluted), cathepsin B (Santa Cruz) (1/1000 diluted), cathepsin D (Santa Cruz) (1/1000 diluted), Bax (Santa Cruz, CA, U.S.A.) (1/1000 diluted), Bcl-2 (Cell Signaling Technology) (1/1000 diluted), LC3 (Abcam, Cambridge, MA, U.S.A.) (1/1000 diluted) and Beclin 1 (Cell Signaling Technology) (1/1000 diluted). They were also administrated peroxidase-labeled goat anti-rabbit IgG (Santa Cruz). Their blots were revealed by the chemiluminescence substrate (ECL Plus) before intensity evaluation. The total proteins were exerted for comparison to evaluate the corresponding targeted proteins’ phosphorylation levels while GAPDH protein was exerted for loading controlling.