My lab received some mice heads that have been (and still are) in PFA for more than 2 years. What do I have to do to be able to extract the brains? And is it possible to freeze this tissue and use it for immunochemistry stainings?
It may be quite difficult to get the optimum result for your intended assay because the heads of the mice were fixed in PFA (More than 2 years is a whole lot) and not the brains which may preclude proper fixation. By freezing, do you mean cryosection and cut at a particular size for IHC? well you can only try and see If you’d get what you want.
There are a lot of unknowns here... but specifically 2years in PFA the brains are well fixed, and antigens are most definitely cross-linked, which will require a minimum of a quality antigen retrieval step. The other question should be addressed prior to starting; were these animals just decapitated and immersion fixed in PFA, or did the previous group perfuse them with a saline/PBS flush followed by PFA? IF they didn't do this it is probably not worth the following efforts because anything you get will be compromised by the presents of blood in the tissues. IF they did the perfusions removing all the blood, you may want to try to carefully extract the brains from the calavera and begin a cryo-protection protocol (sucrose and eventually a Glycerin/Glycol solution). This is a critical step; you can't just freeze the extracted brains without cryoprotection. After which you will be able to section for frozen IHC. At this point you still have questions... depending on your target antigens you are severely limited due to their age in PFA. In other words, there are many pitfalls with this attempt and your outcomes will vary greatly depending on a number of unknown variables. Good luck.
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