Rabbit polyclonal anti caspase 3 antibody

Bilateral Frontal cortices anterior to the bregma were dissected at 5 days after ROSC, and the left cortical tissues were homogenized for western blotting. Total protein lysates were prepared using lysis buffer (Thermo Scientific, Rockford, IL, USA) containing protease inhibitors cocktail (Sigma-Aldrich) and phosSTOP phosphatase Inhibitor Cocktail (Roche, Nutley, NJ, USA). The BCA assay kit (Thermo Fisher Scientific, USA) was used to measure the protein concentration. Samples comprising 20 μg total protein per lane was separated by SDS-PAGE and then transferred to a PVDF membrane. The membranes were blocked with 5% non-fat milk for about 1 h at room temperature and incubated with the following primary antibodies overnight at 4 °C: rabbitpolyclonal anti-caspase-3 antibody (1:1000; Cell Signaling Technology, USA); rabbit polyclonal anti-NF-κB antibody (1:1000Protein-tech, China);α-tubulin (1:5000; Protein-tech, China,). After incubation with secondary antibodies, the immunoreactive bands were developed using enhanced chemiluminescence reagents (Pierce, IL, USA) and was visualized using GeneSnap software version 7.08. The protein amounts were quantified using Image J software and normalized to the density of α-tubulin in the same sample. The results of rats from the different experimental groups were then normalized to the mean values of the corresponding control animals.