Freeze-Substitution Fixative for Yeast

Carry out the following steps wearing protective gloves and working in a fume hood.

1. In a 5-mL conical tube, prepare a stock solution of 5% uranyl acetate by dissolving 0.1 g of uranyl acetate crystals in 2 mL of methanol. Cover the tube with foil and place it on a rocker for ∼10 min at room temperature to dissolve the crystals.
2. Prepare a stock solution of 10% glutaraldehyde (anhydrous EM grade) in acetone (anhydrous EM grade).
3. Transfer 1.25 mL of the 10% glutaraldehyde solution from Step 2 into a 50-mL conical screw-cap tube, and add 1 mL of the 5% uranyl acetate solution from Step 1.
4. Fill the tube to 50 mL with acetone; the final concentrations of the components are 0.25% glutaraldehyde and 0.1% uranyl acetate.
5. Distribute 1-mL aliquots of the fixative into cryovials and freeze them in liquid nitrogen. Store the aliquots in liquid nitrogen.