In a few weeks I will receive a brain of a mouse perfused with PFA.
With fresh tissue I would use PFA after the collection of the tissue, then salin solution, ethanol, xylene and then I would embed in paraffin so that I can produce slides with the microtome.
My question is: How could I make slides from a perfused brain? Do I have to embed it in paraffin anyway? Can I use the microtome?
The major problem is that we do not have a cryostat!
Hi Nicole,
As you mention that you don't have cryostat therefore, you can use paraffin embedding. There is no problem to process and embed mouse brain tissue after PFA perfusion. You can follow routine tissue processing protocol.
It is all depends on what the end point of it. If you just want an H&E on the tissue, then just process it into paraffin block. if there is IHC involves then you need to make sure the antibody also with paraffin embedding tissue. Otherwise, you would need to sucrose cryopreserve the tissue first and then OCT embedding before cryosectioning.
I need slices to perform in situ hybridization so I need 7um slices at least. We thought about the vibrotome but we don't have it either, so the problem would be to find a lab that has it. But if you say that, even if the brain is perfused, I can embed it in paraffin I will try it! For me, it would be the simplest solution
Nicole, double check the IHC protocol if it has been published already. That will inform you as to the best methods. As Vuong mentions above you may have to modify processes depending on the antibody specifications. Often good to practice on the least important samples first. If you are needing to bring samples to a histology laboratory to use the vibrotome I would recommend you talk with them first as they might also paraffin embed your tissues (there is lots of automated machinery for this process now). They may prefer this as they too. I have previously experienced some poor paraffin embedding. so best leave it to specialists and then you will get good quality sections...(also remember you will need charged or other special glass slides>
Nicole, you can proceed with the perfusion fixed brain with your "normal" paraffin embedding procedure. Continue with the step following the fixation step of your fresh tissue. You have to pay attention not to store the tissue to long in the PFA to avoid over-fixation of the tissue. Otherwise the mRNA could degrade. In situ hybridization works very good on perfusion -fixed (e.g. buffered-4% PFA) and paraffin embedded brain. There is no need to go for ISH on vibratome or cryosections. In the latter ones morphology is not as good as in paraffin sections.
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