Isolation buffer

Spinal cord tissue (1 cm with injury epicenter located centrally) was carefully removed and stored at − 80 °C until use for quantitative real-time PCR (qPCR), western blot, and myeloperoxidase (MPO) activity detection.
The spinal cord tissue was harvested and homogenized with isolation buffer (Beyotime Institute of Biotechnology, Shanghai, China) in ice-chilled Dounce homogenizers (1:10, w/v), and then centrifuged at 1000g for 5 min. Supernatants were transferred into another tube, centrifuged at 8000g for 10 min, subsequently removed and centrifuged at 12000g to acquire cytosol fractions. Mitochondria-enriched pellets were resuspended and washed with isolation buffer, subsequently re-pelleted by centrifugation at 1000g for 5 min and 8000g for 10 min. The cytosol fraction was used for determining cytosolic cytochrome c (Cyt C) levels.

The hippocampal tissues were homogenized in ice-cold Dounce homogenizers (1:10, w/v) with isolation buffer (Beyotime Institute of Biotechnology, Shanghai, China) and centrifuged at 1,000× g for 5 min at 4°C. Supernatants were removed and centrifuged at 12,000× g for 10 min at 4°C to obtain pure cytosolic fractions. The mitochondria-enriched pellets were gently resuspended and re-pelleted by centrifugation at 12,000× g for 10 min. The concentrations of mitochondrial protein and cytosolic protein were determined with a Micro BCA protein assay kit (Beyotime Institute of Biotechnology) with bovine serum albumin as the standard.

Hippocampal tissues were homogenized in ice-chilled Dounce homogenizers (1:10, w/v) using isolation buffer (Beyotime Institute of Biotechnology, Shanghai, China) and centrifuged at 1,000 g for 5 min at 4°C. Supernatants were removed and centrifuged at 12,000 g to obtain pure cytosol fractions, which were isolated to determine cytosolic cytochrome C (Cyt C) levels. The mitochondria-enriched pellets were gently resuspended and washed with isolation buffer, then pelleted by centrifugation at 12,000 g for 5 min. Mitochondria were lysed and the protein was determined by the Micro BCA protein assay kit (Beyotime Institute of Biotechnology).