TEM MONOLAYER PROTOCOL

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

Use for very small pellets or if sample is in a plastic container.
  1. Rinse cell monolayers briefly in PBS.
  2. Fix in situ with 2.5% glutaraldehyde in PBS, pH 7.4, for 1 hour at room temperature.
  3. Wash 3 times in PBS buffer (10 minutes each).
  4. Post-fix monolayers for 1 hour at 4° C in 1% OsO4 with 1% potassium ferricyanide.
  5. Wash 3 times in PBS buffer (10 minutes each).
  6. Dehydrate monolayers in a graded series of alcohol (30%, 50%, 70%, and 90% - 10 minutes) with three changes in 100% ethanol (15 minutes each).
  7. Change three times in epon (1 hour each).
  8. Remove last change of epon and invert beam capsules full of resin over relevant areas of the monolayers and polymerize at 37° C overnight and then 48 hours at 60° C.
  9. Pop off the beam capsules and underlying cells from the bottom of the petri dish and section.