Hi guys,
Following perfusion, some of my rat brains were fixed and put in cryoprotectant solution (-20°C) whole as i did not have time to section them. They have been there for around 5 months. I now wish to block, slice, and IF stain for cFos (and some other cell bodies/receptors).
Could anyone recommend some steps to ensure the quality of this staining? I.e. do i need to extensively wash (PBT?) the cryo off, or blot it off with tissue?
Also, are there any issues which i may face? E.g. as they were fixed and cryoprotected whole, would i expect IF to work OK compared to if they were sliced earlier?
Thanks :)
you should be okay. I have experience with muscles and cryoprotectant and the staining always turns out fine. Just soak in PBS (though I am not too paranoid about the cryoprotectant I do wash it). An overnight soak in PBS at 4C won't hurt. If you are really concerned, you could do this with your slices as well. Make sure you flash freeze ( methyl butane). Give one a try and see how it looks!
Sectioning frozen brain tissue is usually not as easy as sectioning most other tissues (also not easy: breast and fat tissues) - I recommend to ask a very experienced technician to prepare thin brain slices at similar quality, so the further steps may have better chances to avoid artefacts, like trapping antibodies under broken parts of the slices...
Frozen samples often not suitable for virology assay because ice crystal could be damaged to virus cell wall and destroyed it so I prefer to use some chemical fixatives such as formalin 10% and Bouin solution for pathology assay and then following by IFAT or IHC examination.
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