Processing hard to penetrate tissues for standard TEM

1. Fixation:
According to tissue
2. Wash:
3x15 min. with correlative buffer @ RT
3. Osmification:
1 or 2% OsO4 in correlative buffer 1-2 hours @RT
4. Wash:
3x15 min. ddH2O @RT
5. Dehydration with ETOH and Propylene Oxide @RT:
30%, 50%, 70%, 85%, 95%, 100% ETOH 1x20 min. each
200 Proof (pure) ETOH 1x20 min.
Pure PO 2x20 min.
6. Infiltration with Epon:
PO:Epon = 1:1 for 1 hr.
PO:Epon = 1:2 overnight
Pure Epon for >1hr.
7. Embedment:
Embed into blocks and cure @45C overnight and @60C 48 hr.