Müller, M., Marti, T., and Kriz, S. 1980. Improved structural preservation by freeze- substitution. Electron Microscopy, Vol. 2, pp. 720–721, Brederoo, P., and De Priester W.(Eds.), North Holland, Amsterdam, The Netherlands.
Lecithin coating of carriers:
Prepare a solution of 2% (w/v) Lecithin (L-α-Phosphatidylcholine from egg yolk, Fluka-61755) in Chloroform. Dip the type A carriers one by one in the Lecithin solution and lay the carriers with the 100 μm deep side up on filter paper. Dip also the type B carriers in the Lecithin solution and lay the carriers with the flat side up on filter paper. The carriers will be covered with a thin layer of Lecithin after the Chloroform has evaporated.
1. Solution A: prepare 10 ml 0.6% (w/v) Uranyl Acetate in anhydrous Methanol (Merck, 1.06012) and next dissolve 200 mg OsO4. Cool down this solution for 30 min at –90° C in the AFS2.
2. Solution B: prepare 10 ml 6% (v/v) Glutaraldehyde in anhydrous Methanol (from 10% (v/v) GA stock in anhydrous Methanol (EMS, 16532)) and cool down this solution for 30 min at –90° C in the AFS2.
3. Cool down 2.0 ml Sarstedt tubes for 30 min at –90° C in the AFS2. Remove the rubber ring from the lids before cooling down the tubes.
4. Cool down a 15 ml glass vial with snap or screw cap for 30 min at –90° C in the AFS2.
5. Pipet equal volumes of precooled solution A and B in the precooled 15 ml glass vial, and mix well by pipetting up and down at least three times.
6. Fill 2.0 ml Sarstedt tubes with 800 μl precooled FS medium
For approximately 50 ml pure Epon add together:
1. 21.5 gram Epoxy embedding medium (Fluka)
2. 25.0 gram Araldite M Hardener 964 (Fluka)
3. 7.0 gram Epoxy embedding medium hardener MNA (Fluka)
Mix well for at least 10 min and add:
4. 0.75 ml Epoxy embedding medium accelerator DMP 30 (Fluka)
Mix well for at least another 10 min.