Postfixation

Osmium Tetroxide (OsO4)

Osmium tetroxide functions as a secondary fixative by reacting with lipids. It is believed that the unsaturated bonds of fatty acids are oxidized by OSO4 and it is reduced to a black metallic osmium (mw-254.2) which is electron dense and adds contrast to biological tissues (secondary stain).

1. Osmium tetroxide penetrates slower than glutaraldehyde (0.5 mm/hr.) and tissue take on a progressively blackened appearance depending upon their lipid content.

2. Osmium tetroxide completely permeabilizes cell membranes and the osmolarity of the fixative vehicle or solute is relatively unimportant.

3. It penetrates tissue blocks in a gradient and in large samples the center of the block may not be as well fixed as the peripheral areas.

4. Overfixation with osmium tetroxide may result in extraction of cell components during dehydration and increases the hardness and brittleness of the tissue. (for most tissues, 1mm blocks, should not be exposed to osmium for less than 0.5 or more than 1.5 hours).

5. Osmium will turn the environment acid and its vehicle should be buffered strongly.