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Microscopy Laboratory Techniques

Tetrahymena Fixation for Transmission Electron Microscopy

  1. Pellet Tetrahymena cells in a clinical centrifuge.
    OPTIONAL: Suspend cells in HNMK (50 mM HEPES, pH 6.9, 36 mM NaCl, 0.1 mM Mg acetate, 1 mM KCl ) for 10-20 min at room temperture. This will remove precipitates present in the proteose peptone and provide cleaner surfaces if cells are processed for SEM.
  2. Pour off most of the medium and suspend cells in a slurry (<1 ml of remaining medium) at the bottom of the tube (be gentle).
  3. Fix by adding 5-10 ml of 2.5% glutaraldehyde in 100 mM HEPES, pH 7.2. Mix gently and let sit at room temperture for 1 hr.
  4. Gently pellet cells in the clinical centrifuge (don't pack cells). Suspend gently in 100 mM NaCacodylate, pH 7.2.
  5. Repeat step 4 three times.
  6. Prepare 0.5-1% OsO4 in NaCacodylate, pH 7.2 and add to the fixed cells - suspend gently and incubate on ice for 30-60 min.
  7. Rinse 3X, ~5 min/rinse, with water (gently pelleting each time, if necessary).
  8. Suspend final washed cells in 0.5 - 1% uranyl acetate (aqueous). Incubate from 30 min to overnight.
  9. Dehydrate cells in acetone (25%, 50%, 75%, 3X100%; 3-10 min/change).
  10. Embed in epon resin. I mix dehydrated cells with 1/3 plastic and 2/3 acetone and let them sit on a rotator for a few hours or, uncovered, overnight in the hood (on the rotator). The next day, I pellet the cells and suspend them in 100% plastic. Suspend cells in a small amount of plastic and then underlay the suspension with 100% plastic. Let the cells sit for several hours and place in house vacuum for ~ 1 hr.

    For final embedding, pellet cells, remove all plastic. Cut the tops off BEEM capsules, drop the capsules in a plastic conical centrifuge tube, and fill BEEM capsules ~half full with plastic. Suspend pelleted cells and lay over fresh plastic. Centrifuge the cells through the plastic. Remove the plastic and add additional plastic to the pelleted cells. Stirr them up a bit. Then centrifuge again (clinical centrifuge, #7, 30 min) to pellet the cells very well. Put tubes at 40 degrees C overnight and raise to 70-80 degrees C for ~24 hrs.

    Polymerize at 40 degrees C overnight and raise to 65-80 degrees for a second day.

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