Hippocampal Fractionation and Mitochondrial Isolation

A particulate and soluble fractions together with a pure-mitochondria fraction were obtained from the CA1 and CA2-4,DG regions of the hippocampi of control and ischemic gerbils. Hippocampi were homogenized in ice-cold isotonic buffer (15 mM Tris/HCl, pH 7.5, 0.25 M sucrose, 1 mM MgCl₂, 1 mM EGTA, 2 mM EDTA, 1 mM PMSF, and 1 mM DTT), prior to centrifugation (1000× g, 10 min, 4 °C). The supernatant was centrifuged at 11,000× g for 20 min at 4 °C to yield a particulate fraction enriched with mitochondria (P2). The pure mitochondrial pellet was obtained after centrifugation of P2 (100,000× g, 30 min, 4 °C) with 12% Ficoll, as described earlier [4 (link),30 (link)]. For Western blot analysis of MPC1 and MPC2 the tissue was homogenized under nondenaturing conditions, in cell lysis buffer containing 20 mM Tris–HCl at pH 7.5, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 μg/mL leupeptin, and 1 mM PMSF (Cell Signaling Technology, Danvers, MA, USA). After homogenization samples were sonicated four times for 5 s and centrifuged 14,000× g at 4 °C for 20 min to obtain clear tissue lysates. The protein concentration was determined using a Modified Lowry Protein Assay Kit (Thermo Scientific, Grand Island, NY, USA).