Quantifying Mitochondrial Changes in Brain Tissue

Brains were removed at 0 h, 24 h, 72 h, and 7 d after I-R insult or sham operation, and a section of the cortex was cut into pieces measuring approximately 1 mm³ and processed as previously described [26] (link). The small blocks were placed in an ice-cold fixative (2% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4) for 5 h. The samples were subsequently washed extensively with 0.1 M cacodylate buffer, post-fixed for 2 h with 2% OsO₄/0.1 M cacodylate buffer, dehydrated in ethanol, block-stained with uranylacetate, and embedded in Epon. Ultrathin sections were collected on copper grids, double stained with uranylacetate and lead citrate, and examined using a Tecnai G2 Transmission Electron Microscopy (FEI, Portland, OR, USA). All analyses were performed in a blinded and non-biased manner. To measure the mitochondrial number, 15 randomly selected areas per animal, which included large neuronal-like nuclei covering approximately one-quarter of the visible image, were imaged at 8200× magnification and counted using minor modifications to a previously described method [21] (link).