Mitochondrial Ultrastructural Analysis

Cells were fixed with freshly prepared fixative containing 2.5% glutaraldehyde in 0.15 M cacodylate buffer. Fixed cells were postfixed in 1% OsO₄ in 0.1 M cacodylate buffer for 1 hour on ice. Cells were stained en bloc with 2%–3% uranyl acetate for 1 hour on ice as described previously (95 (link)). The cells were dehydrated in a graded series of ethanol (20%–100%) on ice followed by 1 wash with 100% ethanol and 2 washes with acetone (15 minutes each) and embedded with Durcupan. Ultrathin (50–60 nm) sections were cut on a Leica UCT ultramicrotome, and picked up on Formvar and carbon-coated copper grids. Sections were stained with 2% uranyl acetate for 5 minutes and Sato’s lead stain for 1 minute. Grids were viewed using a JEOL JEM1400-plus TEM and photographed using a Gatan OneView digital camera with 4k × 4k resolution. Morphometric measurements were made randomly on deidentified samples. The free-hand tool in ImageJ was used to determine mitochondrial area by manually tracing around the mitochondrial outer membrane, as described previously (96 (link), 97 (link)). The area of each crista membrane was also calculated in the same manner. The sum of the areas of the total complement of cristae was then divided by the sum of the mitochondrial area to obtain the cristae density.