All rats of each group were anesthetized with ether and then were decapitated. The brain was removed rapidly and the hippocampus was dissected and put onto a box chilled with ice. After the hippocampal tissue was rinsed with PBS buffer 3 times, the hippocampal tissue was homogenized at 1: 5 (wt/vol) in an ice-cold lysis buffer with protease inhibitor cocktail and phosphatase inhibitor cocktail.
The samples were subjected to SDS-PAGE and were transferred to Polyvinylidene Fluoride (PVDF) membranes. The blots were probed with the following primary antibodies: polyclonal mouse anti β-actin (Sigma), rabbit anti-Akt (Cell Signaling), mouse anti-pAkt (Cell Signaling), rabbit anti-CREB (Cell Signaling), mouse anti-pCREB (Cell Signaling), rabbit anti-PGC1α (Cell Signaling) and rabbit anti-SOD2 (Cell Signaling), monoclonal mouse anti-Bax (Cell Signaling), monoclonal mouse anti-Bcl-2 (Cell Signaling), polyclonal rabbit anti-caspase-3 (Santa Cruz), followed by incubation with species-matched horseradish peroxidase-conjugated secondary antibodies. The blots were developed with a chemiluminescence substrate solution (Pierce) and exposed to X-ray film. The optical density of immunoreactive bands was quantified using the Quantity One software (Bio-Rad).
The samples were subjected to SDS-PAGE and were transferred to Polyvinylidene Fluoride (PVDF) membranes. The blots were probed with the following primary antibodies: polyclonal mouse anti β-actin (Sigma), rabbit anti-Akt (Cell Signaling), mouse anti-pAkt (Cell Signaling), rabbit anti-CREB (Cell Signaling), mouse anti-pCREB (Cell Signaling), rabbit anti-PGC1α (Cell Signaling) and rabbit anti-SOD2 (Cell Signaling), monoclonal mouse anti-Bax (Cell Signaling), monoclonal mouse anti-Bcl-2 (Cell Signaling), polyclonal rabbit anti-caspase-3 (Santa Cruz), followed by incubation with species-matched horseradish peroxidase-conjugated secondary antibodies. The blots were developed with a chemiluminescence substrate solution (Pierce) and exposed to X-ray film. The optical density of immunoreactive bands was quantified using the Quantity One software (Bio-Rad).