Osmium tetroxide is extremely toxic and must be handled with care in a fume hood using the appropriate PPE. Used osmium should be discarded in a labelled plastic container containing ethanol to reduce and neutralise the osmium.
Osmium can precipitate out of solution when in the presence of glutaraldehyde to form small electron dense artefactual deposits. Samples must be washed thoroughly (Step 3) prior to post-fixation (Step 4) to avoid this.
Potassium ferrocyanide reduces osmium, turning the post fixation solution black. Reduced osmium may also prevent the formation of artefactual precipitates. Potassium ferricyanide can be used as an alternative in combination with osmium tetroxide. This solution is a clear, amber colour.
The following steps can be performed with or without the use of a PELCO BioWave® Pro+ Microwave Processing System. A Biowave facilitates infiltration of reagents into tissue and cells, and reduces the processing time.
Post fix the tissue in 1% Osmium tetroxide, 1.5% potassium ferrocyanide in 0.1M cacodylate buffer for 2 hours at room temperature, agitating. Tube or well-plate lids must be well-secured during fixation.
Biowave settings: 3x 120 seconds, 100W, Vacuum on.
A two minute pause step without a solution change must be included in between each interval to prevent overheating of samples.