Principles & Protocols

Protocols - Freeze Substitution

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Instructions for preparing FS solutions

To make up 50 ml of Fixative: REMEMBER! YOU MUST WEAR DOUBLE GLOVES FOR THIS PROCEDURE AND WORK WELL INSIDE IN A FUME HOOD THAT DRAWS WELL. OSMIUM IN ACETONE IS VERY VOLATILE!

Label, with the #2 pencil, about 35 Nalgene 2.0 ml cryovials.
Put vials in vial rack with tops off.
Make up stock solution of 5% uranyl acetate in methanol by adding 0.1 g of uranyl acetate to 2 ml pure methanol, cover with foil and put on rocker to dissolve. It should dissolve in about 10 minutes, but if it doesn't, just let it rock longer. Different vintages of uranyl acetate crystals seem to dissolve at different rates.
Dip a 1 g OsO4 ampule for about 10 seconds into liquid nitrogen to loosen crystals from glass.
Fill 50 ml conical tube with 45 ml pure acetone.
Add 1 g OsO4 crystals to the 45 ml of acetone, cover and mix until completely dissolved.
Add 1 ml 5% uranyl acetate stock to OsO4-acetone, add pure acetone to make 50 ml and mix well.
Use repeater pipettor to dispense 1.5 ml into each cryovial. Work quickly, but not carelessly. Cap cryovials as soon as they are filled.
Add LN2 to Styrafoam box to a level that will cover cryovials in rack.
Slowly lower rack into LN2. Vials must remain upright so fixative will remain in the bottom of the vial.
When completely frozen, transfer vials to LN2 storage device until ready to use.

Sample record sheet:

Equipment operation

Wear oxygen monitor and fill AFS through funnel with liquid nitrogen.
Warm up sample well to 30C for 30 min to remove all moisture.
After heat cycle fill sample well with reagent methanol
Program substitution sequence
- If a sequence is running press pause and hold until you hear three beeps
Begin program and pause until starting temperature is reached
Insert samples into AFS sample well
Begin freeze substitution cycle

Finally

Rinse 3x with dehydrated acetone for 1h each
Infiltrate with resin

-- KdDerr - 02 Sep 2009