FIXATION OF MOUSE TISSUE FOR TRANSMISSION ELECTRON MICROSCOPY
A. Preparation of Tissue
B. Primary Fixation
- Sacrifice mouse by cervical dislocation and remove organs to be fixed (e.g., liver and heart).
- Remove a small piece of each tissue to be fixed, and mince it into cubes no larger than 1 mm on a side. Moistening the tissue with a small volume of saline will help reduce sticking of the tissue to the dish and instruments.
C. Secondary Fixation
- Place the tissue into a vial containing enough fixative to cover the tissue well. Primary fixative = 2% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M cacodylate - 0.1 M sucrose, pH 7.4.
- Let stand at room temperature (RT) for at least 1 hour with occasional agitation. At this point the tissue may be stored overnight or for days at 4° C.
D. En Bloc Staining - optional contrast enhancement
- Rinse tissue with three changes of 0.1 M cacodylate - 0.1 M sucrose buffer, pH 7.4, 5-10 min./change, RT.
- Add enough ice cold 1% OsO4 in 0.1 M cacodylate - 0.1 M sucrose buffer, pH 7.4, to cover the tissue, and let stand on ice or at 4° C for 1 to 2 hr. CAUTION: Work under the hood with osmium.
- Rinse tissue with two changes of 0.1 M cacodylate - 0.1 M sucrose buffer, 5-10 min./change, RT.
- Rinse once with 0.05 M maleate buffer, pH 5.15, 5-10 min., RT°.
- Cover tissue with 2% uranyl magnesium acetate in 0.05 M maleate buffer, pH 5.15, 1 hr., RT°.
- Rinse 2X with 0.05 M maleate buffer, pH 5.15, 5-10 min., RT°.
F. Transition solvent - OPTIONAL (at your own risk) for Spurr's embedding resin
- Rinse 1X with distilled H2O, 5-10 min., RT°.
- Dehydrate stepwise in the following concentrations of ethanol, 5-10 min./change, RT.: 30%, 50%, 70%, 90%, 95%. At this point the tissue may be stored overnight at 4° C.
- Rinse 3X with 100% ethanol, 10 min./change, RT°.
G1. Infiltration and embedding (Spurr's low viscosity resin)
- Continue dehydration with 1:1 propylene oxide / 100% ethanol for 10 minutes.
- Rinse 2X with 100% propylene oxide, 10 min./change, RT°.
- Infiltrate tissue with 1/3 Spurr's (1 part resin: 2 parts ethanol), 15 minutes, RT°.
- Infiltrate tissue with 2/3 Spurr's (2 parts resin: 1 part ethanol), 15 minutes, RT°.
- Two changes of 100% Spurr's resin, 15 min./change.
- Embed in fresh Spurr's resin (less than one day old). Polymerize for 6 to 8 hours at 70° C.
alternative embedding medium:
G2. Infiltration & Embedding (Epon 812, Embed 812, Polybed 812). See also alternate procedure for Spurr's resin below)
- Infiltrate tissue with 1/3 epoxy resin (1 part resin : 2 parts propylene oxide, 15 minutes, RT°.
- Infiltrate tissue with 2/3 epoxy resin (2 parts resin : 1 part propylene oxide), 15 minutes, RT°.
- Two changes of 100% pure resin, 15 min./change, RT°.
- Embed in pure resin. Polymerize for 36 hours at 60° C.