Muscle Processing for Standard TEM

Muscle protocol and flat embedment muscle protocol. Utilize a rotator or shaker if possible for all steps.

1. Fixation:
According to tissue
2. Wash:
2x15 min. 0.1M Tris buffer @RT
2x15 min. isoosmotic buffer @RT
3. Osmification:
1% OsO4 in 0.1M PB 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 1x15-20 min. each
200 Proof (pure) ETOH 1x15-20 min.
Pure PO 2x15-20 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.

0.18M Phosphate Buffer (muscle)

Isoosmotic Phosphate Buffer (Muscle)

0.1M Tris Wash (Muscle)

0.4M Phosphate buffer Stock (Muscle)

Mouse Glutaraldehyde Fixative (muscle)

1% OsO4 in 0.1M PB for Muscle