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.