Fixation:

1.       Fix 1 mm tissue blocks in 4% formaldehyde and 1% glutaraldehyde in 0.1 M PB (pH 7.4) for at least 2 hours to overnight.

2.       Immerse in 8% (0.2M) sucrose in 0.1 M PB 3x15min or overnight.

3.       Post-fix in 1% osmium tetroxide in 0.1 M PB 1 hour.

Dehydration:

4.       50% ethanol  15min

5.       70% Ethanol  15min

6.       95% Ethanol  15min

7.       100% Ethanol  2x15min

8.       100% Propylene oxide  2x15min

9.       1:1 Epon and Propylene Oxide for1-2 hour.

10.    2:1 Epon:Propylene Oxide overnight in dessicator with top off.

Embedding:

11.    Embed in beam capsules.

12.    Bake in 60 C oven for 48 hours.

Sectioning:

13.    Semithin section and Toluidine blue stain for 2-5 min

14.    Observe sections under microscope for precise location to cut for ultrathin sections