TEM TISSUE RUN UP PROTOCOL

Gloves must be worn throughout the procedure. All work should be done in a fume hood.
  1. Fix tissue in 2.5% glutaraldehyde in 0.1 M PBS (pH 7.4) for 10 minutes.
  2. Cut up tissue into small blocks (1mm³) and continue fixing for at least 50 minutes.
  3. Wash in 3 changes 0.1 M PBS for 15 minutes each.
  4. Post fix specimens in 1% Osmium tetroxide containing 1% potassium ferricyanide for 1 hr.
  5. Wash in 3 changes 0.1 M PBS for 15 minutes each.
  6. Dehydrate in graded series of alcohol (in PBS) for 15 minutes each:
    1. 30% ethanol
    2. 50% ethanol
    3. 70% ethanol
    4. 90% ethanol
    5. 100% ethanol x 3
  7. Dehydrate further in two 10 minute changes of propylene oxide. *
  8. Infiltrate with a 1:1 mix of propylene oxide and epon for 1 – 3 hrs or overnight.
  9. Infiltrate with pure epon overnight at 4° C or 1 hour at room temperature.
  10. Continue infiltration with three 1 hour changes of epon.
  11. Embed in pure epon at 37° C for 24 hours.
  12. Cure for 48 hours at 60° C.

*DO NOT USE IF FOR VERY SMALL PELLETS OR IF SAMPLE IS IN A PLASTIC CONTAINER. USE THE MONOLAYER PROTOCOL.: