4.4.1. Isolation of Rat Brain Mitochondria

The whole brain was rapidly removed and freed from blood and vessels. Rat brain mitochondria were isolated by differential centrifugation according to the method of Sims [64]. The tissue was finely minced with scissors in a small amount of isolation buffer (0.32 M sucrose, 20 mM HEPES, 1 mM EGTA, 10 mM Tris/ HCI, pH 7.4) and washed three times with this buffer. The tissue in isolation buffer (l0%, wt/vol) was homogenized. The homogenate was centrifuged at 1330× g for 3 min and the supernatant was carefully decanted and the pellet resuspended in half of the original volume. Subsequently, homogenate was re-centrifuged as above, the supernatant was retained, and the pellet discarded. The pooled supernatant was centrifuged at 20,200× g for 10 min. The decanted supernatant was discarded, and the pellet resuspended in 15% Percoll. Tubes were centrifuged for 5 min at 21,700× g. Three major bands of material were obtained, and the material banding near the interface of the lower two Percoll layers was diluted 1:4 by gently mixing with isolation buffer and then centrifuged at 16,700× g for 10 min. The supernatant was removed and the material gently resuspended. Fatty acid-free bovine serum albumin (10 mg/mL) was added, and the mixture was diluted with isolation buffer (final volume 3 mL). After centrifugation at 6900× g for 10 min, the supernatant was rapidly decanted, and the pellet gently re-suspended in an isolation buffer without EGTA. This fraction was stored on ice for further investigations.