I am not familiar with the fixation process that the brains have been subjected to you. However, we have placed them in -80 C to ready for sectioning, but the tissue falls apart. Any suggestions?
Hi,
the hystological way to go is to use OCT compounds in order to prepare the tissue for cutting.
If you prepare the tissue for mass spec imaging purposes, OCT should be avoided (used only to fix a small portion of the tissue to the cryostat holder).
No OCT means that the tissue sample will need to be cut at higher thickness. In general, 10-12 um is a good thickness to start with. Lower than that, you need skills and experience (and even then, the tissue might just fall apart).
Did you use any fixant (ethanol, formalin etc..)?
Thanks for your answer. We do use OCT to adhere the block of tissue to the chuck, and then pack with dry ice. I believe the tissue is soaked in formalin, but it is older tissue, so not sure of the protocol.
If your tissue didn't have fix in it, but just cryoprotectant such as 30% sucrose, it might allow growth of fungus. Did you see any fungal mats on your tissue or in the solution? If you smelled formaldehyde, it could have been in either plain fix or a mix of fix and cryoprotectant.
In answer to your question, I'd say there's no reason why you couldn't go back and add fix to the tissue, so long as the cryoprotectant it's in hasn't become too viscous (due to water evaporation, which can happen to old samples). But that would probably help only if the tissue hadn't been fixed the first time around, perhaps due to some need to preserve some protein structure. (If you have indeed smelled formaldehyde from it, that'd rule out this possibility.)
However, I would recommend finding out how the tissue was prepared, if possible. :-)
First of all i want you to understand the process of Fixation; arresting autolytic Change that may set it, so Long as the tissue is no longer alive. If you have fixed the brain tissue and if certain cells or portions are not well fixed, then you will not get the desired results. I suggest you cut again aspect of the brain, since the ones you cut for the cryostat are just small pieces.
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