Ultrastructural Analysis of Mitochondria

Targeted fresh tissues were selected to minimize the mechanical damage, then cut with a sharp blade and harvested quickly within 1–3 min. The 1-mm³ tissue blocks were transferred into an EP tube with fresh TEM fixative for further fixation at 4 °C for preservation and transportation. The tissues were washed using 0.1 M PB (pH 7.4) three times for 15 min. Tissues were further fixed with 1% OsO4 in 0.1 M PB (pH 7.4) for 2 h at room temperature, followed by dehydration at room temperature. The resin blocks were cut into 60–80-nm sections on an ultra microtome, and the tissues were fished onto 150 mesh cuprum grids with a formvar film. Images were captured using a transmission electron microscope (HITACHI; HT7800/HT7700). Image J was used on transmission electron microscopic images to observe individual mitochondrial morphology, as described previously [27 (link)].