Routine Morphology Protocol

1. Fix tissue (2% gluteraldehyde, + 3% paraformaldehyde, + 5% Sucrose) in Sodium Cacodylate buffer (pH7.4)

2. Wash 0.1M cacodylate buffer + 7.5% sucrose.

3. Post fix Palade's Osmium one hour on ice in light tight container.

Palade's Osmium
1% osmium
Acetate veronal
0.01N HCl
ddH2O

4. Rinse 1X Kellenbergers UA. Then incubate 2-4 hours in Kellenberger's (overnight)

Acetate Veronal
HCl
Uranyl Acetate
H20

5. Quick rinse in cold 50% ETOH

6. Dehydrate with graded series of ethanol (75,90,95,100,100 100) 5 min each.
Place in a tube with 50% (100%) ETOH and 50% Propylene oxide 5 min.
Place in a tube with 100% Propylene oxide 5-10 min.

7. Place in a tube with 50%PO + 50% Epoxy resin. Let sit on rotator or under
vacuum overnight.

8. Replace epon with fresh 2X, leave under vacuum.

9. Place tissue in mold and cure at 60o overnight.

10. Start cutting.

11. Pick up sections on formvar coated grids, stain with UA (20 min) and Lead citrate (4-6min)

12. Scope.