Beem capsules with conical tip

Extracted mitochondria were treated based on (Unger et al, 2017) (link), but fixed in suspension using 1.5% glutaraldehyde, 3% formaldehyde, and 2.5% sucrose in 0.1M sodium cacodylate buffer o/n at 4°C. Mitochondria were spun down into a pellet at 13,000g in a 1.5-ml microfuge tube. The fragile pellet was washed carefully three times with ddH₂O and postfixed with 1% osmium tetroxide for 1 h at 4°C. The pellet was washed four times with ddH₂O and incubated in 0.5% uranyl acetate overnight at 4°C. The pellet was washed three times in ddH₂O and embedded in 2% low-melting agarose, which was cut into small pieces of 1-mm edge length using a razor blade. Agar pieces were dehydrated for 15 min using ascending ethanol concentrations of 50%, 70%, 90%, 2× 100%, and 2× propylene oxide at 4°C. Pieces were infiltrated with Epon/propylene oxide 1:1 overnight at 4°C and pure Epon for 6 h at RT and embedded into BEEM capsules with conical tip (#69913-01; Science Services) and cured for 48 h at 60°C. Images were acquired using a OneView 4K camera (Gatan) mounted on a Jem-2100Plus (Jeol) transmission electron microscope operating at 200 kV. Large montages of 100 images were acquired using SerialEM (Mastronarde, 2003 (link)).