Fixation:

1.       Fix cell suspension or free cells in 4% formaldehyde and 1% glutaraldehyde in 0.1 M PB (pH 7.4) by mixing equal volume of fixative and cell suspension.

2.       Transfer cells to centrifuge tube and spin for 10 minutes. A nice, tight pellet will be formed (Re-spin if necessary during processing). Carefully pipette off fixative. Add fresh fixative for at least 2 hours or overnight.

3.       Pipette off fixative. Replace with 8% (0.2M) sucrose in 0.1 M PB 3x15 minutes or overnight at 4 C (Samples can be kept in sucrose for a long time).

4.       Post-fix with 1% OsO₄ in 0.1 M PB for 1 hour.

5.       Pipette off OsO₄, Rinse in 0.1 M PB 3x10 minutes.

Dehydration:

1.       50% ethanol  15min

2.       70% Ethanol  15min

3.       95% Ethanol  15min

4.       100% Ethanol  2x15min

5.       100% Propylene oxide  2x15min

6.       1:1 Epon and Propylene Oxide for1-2 hour.

7.       2:1 Epon:Propylene Oxide overnight in dessicator with top off.

Embedding:

8.       Embed in Beam capsules.

9.       60 C oven for 48 hours.