Freeze-Substitution Fixative

doi:10.1101/pdb.rec069435

Freeze-Substitution Fixative

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MATERIALS

Reagents

Acetone, pure

Glutaraldehyde, anhydrous EM grade (10% in acetone) (Electron Microscopy Sciences 16530)

Crystalline osmium tetroxide (OsO₄; 1 g ampule) may be used in place of the glutaraldehyde.

Methanol, anhydrous

Uranyl acetate crystals

The uranyl acetate in this fixative helps with membrane contrast during electron microscopy.

Equipment

Aluminum foil

Bath sonicator

Conical tube with screw cap (50 mL)

Cryovial rack (Nalgene)

Cryovials (2 mL) (Nalgene)

The catalog states that these cryovials should not be immersed in liquid nitrogen (LN₂), but we have been using them for years without problems. Alternatively, buy vials specifically to use with LN₂.

Fume hood

Gloves

LN₂ in 4-L Dewar flask

LN₂ storage device

Pencil (#2)

Repeater pipettor to dispense up to 2-mL aliquots

Styrofoam box (large enough to accept Nalgene cryovial rack)

Vial rocker

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METHOD

This method is for the preparation of 50 mL of fixative.

Important: WEAR DOUBLE GLOVES FOR THIS PROCEDURE AND WORK WELL INSIDE A CHEMICAL FUME HOOD THAT DRAWS WELL. OSMIUM IN ACETONE IS VERY VOLATILE!

1. Use a #2 pencil to label ∼35 Nalgene 2.0-mL cryovials as 2.1-Os-UA (which stands for 2% osmium tetroxide plus 0.1% uranyl acetate). If making a glutaraldehyde fixative, label the vial 0.2.1-G-UA (which stands for 0.2% glutaraldehyde plus 0.1% uranyl acetate). If using a freeze-substitution solvent other than acetone, indicate that also.
2. Put vials in vial rack with tops off.
3. Make up a stock solution of 5% uranyl acetate in methanol by adding 0.1 g of uranyl acetate to 2 mL of pure methanol, cover with aluminum foil, and put on rocker to dissolve (∼10 min). If it has not dissolved after 10 min, let it rock longer.
- Different vintages of uranyl acetate crystals seem to dissolve at different rates.
4. Sonicate an OsO₄ ampule (1 g) for 10 sec to loosen crystals from glass.
5. Fill a 50-mL conical tube with 45 mL of pure acetone.
6. Add 1 g of OsO₄ crystals (or 1 mL of a 10% solution of glutaraldehyde in acetone) to the 45 mL of acetone, cover, and mix until completely dissolved.
7. Add 1 mL of 5% uranyl acetate stock from Step 3 to OsO₄–acetone (or glutaraldehyde–acetone) from Step 6. Add pure acetone to make 50 mL and mix well.
8. Use a repeater pipettor to dispense 1.5 mL into each cryovial. Work quickly, but not carelessly. Cap cryovials as soon as they are filled.
9. Add LN₂ to a Styrofoam box to a level that will cover the cryovials in the rack.
10. Slowly lower the rack into the LN₂.
- Vials must remain upright so that fixative will remain in the bottom of the vial.
11. When completely frozen, transfer vials to an LN₂ storage device until ready to use.