Optimized Transmission Electron Microscopy of Mitochondria

HCT116 cells were grown on round coverslips in a 12-well tissue culture dish. Cells were prepared using a protocol optimized to preserve mitochondrial structure similar to the protocol used by Kim and colleagues^{69 (link)}. Briefly, cells were fixed in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 5 min at room temperature, then continued fixation for 30 minutes on ice. The wash buffer, post-fix, water and uranyl acetate for the following steps were kept ice-cold to allow for optimal mitochondrial preservation. A wash buffer containing 0.1 M sodium cacodylate and 3 mM calcium chloride was used for a series of 5 washes. Washes were followed by a 30 minute post-fixation on ice. The post-fix was comprised of 1% osmium tetroxide, 0.8% potassium ferrocyanide, and 3 mM calcium chloride. The cells were then washed with distilled water and stained with 2% uranyl acetate overnight. The following day, cells were dehydrated, infiltrated and embedded in Epon epoxy and sectioned at 60–90 nm. Sections were imaged on a Phillips CM120 transmission electron microscope (FEI Co., Hillsboro, OR). Measurements of mitochondrial area were taken from round sections, since we interpreted these as being truer to cross-sectional area and reduced bias from longitudinal sections. Area was measured using ImageJ (v1.42q).