2.16. Western Blot Analysis of COX-2, iNOS, Bcl2, Bax, Cleaved Caspase-3 and 9, and Cytochrome-C

The striatal tissues from rat’s brain of each group were used to prepare the homogenate in the RIPA buffer (Merck Millipore, Burlington, MA, USA, catalog number-20188) and cytoplasmic fractions was obtained. Using mitochondrial isolation kit purchased from Abcam, Cambridge, MA, USA (catalog number-110168), and the mitochondrial fraction of the striatal tissues was prepared. The cytoplasmic or mitochondrial fractions from each striatal sample with equal amounts of protein (35 μg) were separated by gel electrophoresis employing SDS-polyacrylamide (10–12%). Then after, the proteins were transferred onto the PVDF membrane and incubated with specific primary antibodies polyclonal rabbit anti-iNOS (1:1000), anti-COX-2 (1:1000), anti-Bax (1:1000), anti-Bcl-2 (1:500), anti-cleaved caspase-3 (1:500), anti-cleaved caspase-9 (1:500), anti-cytochrome-C (1:1000), anti-VDAC (1:2000) and anti-β-actin (1:2000) and kept overnight at 4 °C on shaker for mixing. The PVDF membranes were washed and incubated for 1 h at room temperature with their corresponding secondary antibodies (anti-rabbit or anti-mouse IgG) (sc-2005 and sc-2004) conjugated to horseradish peroxidase. Using an enhanced chemiluminescence pico kit (Thermo Fisher Scientific, Rockford, IL, USA, catalog number: 34580), the protein bands developed were visualized and their intensity was determined by densitometry and quantified with Image J software.