2.8. Determination of expression of caspase-3, Bax and Bcl-2 by Western blot

Injection of zoletil^® (50 mg/kg, i.p.) sacrificed rats for biochemical studies. Brain tissues were quickly removed, and the cerebral cortex and hippocampus were separated on the ice. A 10% homogenate was prepared in lysis buffer, centrifuged at 12,000 (rpm) for 30 min at 4°C. Use the BCA protein assay kit to determine the samples protein concentration. Seventy mg protein was separated on 10% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were incubated for one hr with 5% dry skim milk in TBST buffer at room temperature to block non-specific binding, then with the anti-Caspase-3, anti-Bax, anti- Bcl-2, anti-β-actin antibodies. Later, membranes were incubated with alkaline-phosphatase-conjugated secondary antibody for one hour at room temperature; bands visualized with chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate in the presence of nitroblue tetrazolium.