SEM 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 (8mm³) continue fixing for not more than 50 minutes.
  3. Wash thoroughly in 3 changes 0.1 M PBS for 15 minutes each.
  4. Fix tissue in 1% OsO4 in 0.1 M PBS for 60 minutes.
  5. Wash thoroughly 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. Critical point dry specimen.
  8. Mount on studs.
  9. Sputter coat specimen.
  10. Store in desiccator.