Dear all,
I would ask if there are any advantages/way to use a freeze substitution method to embed samples for transmission electron microscopy in just chemical fixed samples, instead of using it in cryo-fixed high pressure freezed samples.
Many thanks
Francesco
Good question. There are benefits of processing samples at lower temperatures, for instance less extraction during dehydration. A common technique for non-frozen samples is progressive lowering of temperature (PLT).
Unfortunately, one can not simply transfer a non-frozen sample into a cold FS solution, since the sample would be damaged in the subsequent freezing. Which means, the sample will have to be frozen (or at the least protected from freeze damage) in advance. As far as I'm aware, known cryo protectants are not compatible with freeze substitution.
Hello,
Maybe you can think about the Tokuyasu method for cryosectionning and immunolabelling.
- Sample is chemically fixed (then embedded in 12% gelatin solution)
- water in the sample is removed by immersion in 80% sucrose solution
- sample is frozen in LN2.
then frozen sample is cut with a cryoultramicrotome.
In your case, you could start FS from the frozen sample.
Nicolas Barois, in my experience, the sucrose embedding is not suitable for FS.
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