Generic Fixation for Electron Microscopy

Solutions:

100 mM PIPES, pH 7.0-7.4 or 50 mM Cacodylate, pH 7.4
2 mM MgSO₄
0.25 M sucrose
1-2.5% glutaraldehyde

50 mM Cacodylate, pH 7.4 (note: cacodylate contains arsenic, so handle carefully)
1% OsO₄

1% Uranyl acetate in water

Osmium tetroxide and glutaraldehyde can be purchased from the EM lab.

Procedure:

Safety notes: Glutaraldehyde and osmium tetroxide fix cells by cross-linking their proteins via the amine groups (glutaraldehyde) or their phospholipids (osmium). They are harmful to living cells and you should avoid exposure to them. I prefer to use them in a fume hood. Fixatives are volatile and can fix any cells they contact - other cells in the lab or in respiratory epithelium, corneas, epithelial cells on your hand, etc. The good thing is that the fixatives cannot penetrate tissue more than a millimeter or two, so exposure to them rarely causes any permanent damage.

The plastics used to embed the tissue are more dangerous than the fixatives and many of the components of the plastic have been shown to cause cancer in rats or mice. During the embedding process the plastics are dissolved in solvent that can easily carry the plastic into your tissues and through any plastic gloves you may wear. In contrast to fixatives, whose actions are immediate and apparent, the consequences of exposure to the plastic resins are not apparent for years. Please be careful with the plastic resins prior to polymerization into hard blocks. Plastic-containing waste solutions should be kept in a container in the EM lab.

1. Fix tissue by immersion in buffered glutaraldehyde at room temp. Fix for a minimum of 30 - 60 min. Cells can be left in this solution for days if desired.

2. Rinse tissue out of glutaraldehyde and into 50 mM Cacodylate, pH 7.4. Generally 2-4 washes of 10 min/wash is plenty to remove most of the glutaraldehyde.

3. Postfix tissue in the osmium tetroxide solution for 30-60 min. This can be done at room temperature or at 4ûC (on ice).

4. Rinse the tissue in water and stain with 1% uranyl acetate for 30 min to overnight. This helps stain the tissue and seems to help to preserve some structures. If you happen to postfix with osmium tetroxide in phosphate buffer (which is commonly done), be certain to wash well with water. Uranyl acetate + phosphate forms uranyl phosphate, which forms large electron-dense crystals that will ruin the beauty of your tissue.

5. Dehydrate the tissue by immersion in increasing concentrations of acetone or ethanol ( I prefer acetone). Try 25% acetone/water, 50% acetone/water, 75% acetone/water/ and at least 2-3 changes in 100% acetone. Times vary but 2-10 min/acetone solution is fine.

6. Prepare the plastic mixture. The plastics used vary from one investigator to another. One of the most popular and most stable resins is Epon.

   Another popular resin is Spurr resin, mixed as follows:
   

   VCD 		5 g DER 736 	3 g NSA 		13 g DMAE 		0.2 g
     Mix well and transfer to scintillation vials for storage.

7. Mix 1/3 plastic with 2/3 acetone (by volume) and add tissue to the plastic. Let sit for 1-2 hrs or overnight (with the container top removed to let acetone gradually evaporate).

8. Transfer to 2/3 plastic + 1/3 acetone (by volume) for 2-3 hrs.

9. Transfer to 100% plastic and let sit for ~1 hr.

10. Remove old plastic and add fresh plastic to the cells in an appropriate mold.Label the sample, and put in an embedding oven at 60-80 degrees for 1-2 days. By that time the blocks should be polymerized and ready for sectioning.

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