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Microscopy Laboratory Techniques

Chlamydomonas Fixation for Transmission Electron Microscopy

Solutions:

Chlamydomonas culture medium + 2% glutaraldehyde (5 ml medium + 0.9 ml 25% glutaraldehyde

100 mM sodium cacodylate, pH 7.2

1% glutaraldehyde in 100 mM sodium cacodylate, pH 7.2

1% OsO4 in 100 mM sodium cacodylate

Method:

  1. Dilute cells suspended in culture medium into an equal volume of 2% glutaraldehyde in culture medium. Transfer cells to a 15 ml conical polypropylene centrifuge tube. Fix for 15-20 min, room temp.
  2. Gently pellet cells in the clinical centrifuge (2 min setting #6).
  3. Remove supernatant and gently suspend cells in 1% glutaraldehyde in 100 mM sodium cacodylate. Fix 1 hour at room temperature and overnight at room temp 4 degrees C.
  4. Gently pellet cells, remove supernatant, and suspend in 100 mM sodium cacodylate. Repeat this for a total of 4 washes, 2-5 min each wash.
  5. Suspend cells in 1% OsO4 in 100 mM sodium cacodylate and fix for 1 hour.
  6. Gently pellet cells and resuspend in distilled water. Wash 2-3 times with water.
  7. Suspend cells in 1% uranyl acetate in water. Stain 1 hr to overnight at room temp.
  8. Dehydrate cells in acetone. I usually suspend cells in 25% acetone:75% water for ~5 min, let the cells settle or gently centrifuge to lightly pellet cells. Remove the supernate and suspend cells in 50:50 acetone:water for 5 min. Repeat washes with 75%, and two 100% acetone changes.
  9. Embed in Epon resin.

Suspend cells in 1/3 plastic:2/3 acetone and leave the tube uncovered overnight in the hood. If possible, put the tubes on a rotary shaker. The acetone will gradually evaporate and increase the concentration of plastic.

The next day gently pellet the cells, remove the acetone:plastic mix and add fresh 100% plastic. I generally underlay the cells with plastic, let them sit for ~30 min, and place the tubes in a vacuum chamber - pumped by house vacuum. Cells will settle to the bottom of the tube within 1-2 hrs. Pellet the cells in the clinical centrifuge and remove the plastic above the cells.

Put BEEM capsules in the bottom of plastic or glass centrifuge tubes, fill about half full with fresh plastic, and overlay with cells from the above pellets. Spin at top speed for ~ 10 min. Put a label in the top of the capsule and put the tubes in the oven.

Polymerize at 40 degrees C overnight and raise to 65-80 degrees C for a second day or just put at 65 degrees C for 1-2 days.


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