I was thinking of fixing tissues/cells with osmium tetraoxide, since it has been described to reduce the autofluorescence of tissue preparations. However, I was strongly discouraged by the warnings of high toxicity, and stories of buildings being evacuated after a spill. Any experience or tips to deal with the solution in a safe way?
It is reported as a fixative, in which the autofluorescence of parafin and pfa fixed samples disappear.
Yes, it is a toxic chemical but with the availability of a good fume hood and proper storage, the use of OsO4 should not pose a problem. I have been using this fixative for electron microscopy sample preparation for about 40 years. Please find attached the standard preparation protocol for OsO4 as used in my laboratory.
Dear Patchamuthu,
I have read it carefully. I really thank you for such a descriptive protocol. I can see that using your method, the risk of poisoning is almost nothing! That will make my people in the lab really happy!
Just a question about the last part: Once you want to use the solution, it has to be mixed with a proportion 1:1 with 0,2 M Phosphate buffer. Could you tell me which pH does the buffer need to have?
Thanks again!
Dear Luisa,
We routinely use a pH of 7.2 for general EM tissue processing, but I have noted in publications where researchers do use other pHs to suit the type of tissue that they are processing. For example, if the organism/tissue is from an acidic environment, the pH chosen would be to match this pH. Cheers.
Dear Luisa,
I would like to add another tip on this subject. Keep a small container of " full cream milk powder" in your lab. If there happens to be a OsO4 spill, just drop some of this milk powder on the spill, wait for a while, and clean the spill, wearing gloves. (The idea is to react the OsO4 with the lipid components of the milk powder, thereby, weakening the potency of the OsO4, before cleaning the spill with the milk powder). Cheers.
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