Anila Syed wrote:

My question is: I am embedding corpus callosum in epoxy resin in order to quantify the fibres. I will stain with Toluidine Blue.I am going to fix in glutaraldehyde overnight at 4 deg C. But would I also need to use osmium tetroxide? My reasoning is that it will help to stabilise the lipids in the myelin sheaths and therefore give me a much clearer picture of what is going on.
 This is only for LM and will not go to the EM at all.

    The osmium will not only stabilize the lipids in the myelin, it will act as a stain giving more contrast. It will also make the tissue easier to infiltrate with epoxy be virtue of the action of osmium on membranes. The tissue will also be much easier to see in the resin, especially if you are working with small pieces, if it is osmicated. Of course, osmium does not penetrate tissue very well so unless your tissues are thin (2 mm or less, ideally 1 mm), the center of the tissue may not see the osmium. And. as someone else mentioned, osmium must be used under a "hood", the vapors are toxic and will damage the cornea of your eyes. Osmium is also very expensive due in part to the protective packaging required. The crystals can be dissolved but it does take several days.
    On the other hand, depending on the epoxy you are using, the toluidine blue may look "nicer" without osmium.
    You might want to look at Gerrits et al., J. Neuroscience Methods 45:99-105, 1992. They used Sudan Black B to stain GMA-embedded sections of spinal cord. Beautiful results!

Geoff
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Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029
mcauliff@umdnj.edu
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