Basic Tissue Processing for TEM

Adapted and Prepared by Jeannie Mui

Routine TEM tissue processing usually takes three days, with an additional two days for polymerization.

Day 1

1. Tissue processing must be performed in a glass vial.
2. The type of fixation is tissue-dependent.
3. Wash the tissue with 0.1M sodium cacodylate buffer for 3 x 10 minutes
4. Post-fix the tissue with 1% aqueous osmium tetroxide + 1.5% aqueous potassium ferrocyanide for 2 hours at 4^oC.
5. Wash the tissue in ddH2O for 3 x 5 min.
6. For FIB-SEM, it is recommended to en-bloc stain with 2% aqueous uranyl acetate.
7. Wash the tissue in ddH2O for 3 x 5 min.

Day 2

8. Dehydrate in a graded acetone:ddH20 series of 30%, 50%, 70%, 80%, 90% and 3x100% acetone for 10 minutes (size dependent) each to minimize tissue shrinkage,
9. Infiltrate the tissue with 1:1 Epon:acetone mixture on a rotator overnight.
10. Infiltrate the tissue with a 2:1 Epon:acetone mixture on a rotator for 6 to 8 hours, and then infiltrate with a 3:1 Epon:acetone mixture on a rotator overnight.

Day 3

11. Infiltrate in 100% Epon on a rotator for 2 hours.
12. Remove the cap from the glass vial, and leave the tissue in 100% Epon under vacuum for 2 hours.
13. Embed the tissue in an embedding mould. Add a paper label with the sample ID to the embedding mould.
14. Polymerize the tissue in the oven at 58 to 60^oC for 48 hours.