SEM PROTOCOL

Gloves must be worn throughout the procedure. All work should be done in a fume hood.

Fix tissue in 2.5% glutaraldehyde in 0.1 M PBS (pH 7.4) for 10 minutes. Cut up tissue into small blocks (8mm³) continue fixing for not more than 50 minutes. Wash thoroughly in 3 changes 0.1 M PBS for 15 minutes each. Fix tissue in 1% OsO4 in 0.1 M PBS for 60 minutes. Wash thoroughly in 3 changes 0.1 M PBS for 15 minutes each. Dehydrate in graded series of alcohol (in PBS) for 15 minutes each: 30% ethanol 50% ethanol 70% ethanol 90% ethanol 100% ethanol x 3 Critical point dry specimen. Mount on studs. Sputter coat specimen. Store in desiccator.