Western blotting
The hippocampi were isolated and snap frozen in liquid nitrogen. 20-30 mg of tissue were lysed in RIPA buffer followed by ultrasonic homogenization (sonics VCX750, 30% amplitude, 3 pulses of 7 sec each at 4°C). Samples were centrifuged at 14000 g for 20 minutes at 4°C and the supernatants were collected. A total of 40 µg protein was loaded onto an SDS-PAGE gel for separation and transfer to PVDF membrane. Blots were blocked by 5% non-fat dry milk for 1 hour and overnight incubation at 4 °C with anti-ND5, and anti-ND6 antibodies. Blots were further incubated with secondary antibody before ECL detection. The blots were quantified using ImageJ software.