We have mouse brains that have been sitting in PFA for a couple of months, and we now wish to paraffin-embed these PFA-fixed mouse brains for immunofluorescence microscopy. We have an automatic tissue processor, but I have been told that the brain is somewhat more fragile compared to other organs, and to be careful. Any pointers?
Thanks in advance
Brain tissue can be more "fragile" and we typically would dehydrate by hand (i.e. move cassettes in increasing gradients of ETOH to 1:1 xylene and 100% xylene to melted wax then embed), in place of an automated tissue processer. I will not say that the processor is too rough, but I would test on a unimportant brain before committing the lot. Importantly, the duration in which you expose the tissues is based on a couple of points: 1st if you're doing whole brain or blocked sections (also note the thickness of your section), 2nd how many intermediary steps i.e. bigger jump in concentration = more time in the gradient. Once the brains are in wax they are essentially preserved indefinitely and can be sliced at anytime. Another caveat is the current storage of brains in "PFA for a couple of months" this may present a significant problem for fluorescence, over-fixation can be a problem for fluorescent tagged antibodies, less so with HRP. This also assumes that the brain tissues were profused sufficiently and there is no blood remaining as blood will auto-fluoresce.
I agree with Jeffrey. It bothers me that you store tissue in PFA for months. Normally tissue is stored in PFA at 4C for a day or less depending upon the thickness of the issue. That will affect immunohistochemistry. Next time you can make blocks and than blocks can be stored for ever. If you have done perfusion of animal or not that is critical question too. Other suggestions to process tissue by hand are good since the tissue will not get any damage
Jeffrey P Cheng Thanks for the advice. I intend to process one brain hemisphere at a time for later sagittal sectioning, with each mouse brain hemisphere measuring approx. 4mm thick x 14mm long. Would you have a good estimated time for each dehydration, xylene and wax step? I am relatively new to using this technique with brain.
Our two day mouse brain embedding for 4mm sections is the following:
70% ETOH overnight, 80% ETOH 30min, 95% ETOH 2 times @45min each, 100% ETOH 2 times @45min each; 100% Xylene 2 times @45min each; paraffin-1 45min @ ~55C (should be clear and fully melted), paraffin-2 45min ~55C, paraffin-3 45min ~55C; embed.
Also we typically see about 20% shrinkage from hydrated to dehydrated sections measured prior to 70% and again before placement into wax.
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