Dear all
We accidentally forget to fix our tissue before paraffin embedding and sectioning. I know that post-fixation is done on cryosection using aceton. Is this possible on paraffin embedded tissue as well and if yes can I use aceton as well or do I use 4% PFA (what we normally do for fixation of tissue). In practice, i) do you rinse the whole section in it or do you add a dropplet on the section? ii) how long does it need to stay on?
Thank you in advance for your tips and tricks.
Kind regards
Lisanne
Hi Mohamed Mahdy
Thank you for your reply, indeed I will try new samples.
Kind regards
Dear Lisanna,
post-fixation of material already embedded in paraffin will not help to get a better
morphology. The embedding process (dehydration, etc.) has certainly more or less completely destroyed your tissue. Just try new samples and fix them carefully.
Kind regards,
Fred
I agree with Fred.
Just to put a little more detail to it, the formalin fixative creates new bonds between the proteins to hold the structure of the tissues. The first step of processing involves replacing water with alcohol, the water is polar causing hydrophobic/hydrophilic tensions to form for specific structures (i.e. phospholipid bilayer ). The non-polar alcohol does not maintain the hydrophobic/hydrophilic tensions and the proteins are able to separate.
The movement of proteins will be further aided by the heat and pressure often used to assist penetration.
So unfortunately there is not repairing the damage, also I would recommend replacing all your reagents on the tissue processor as they are likely to be have a lot of degraded cells in them.
Post fixation works with cryosectioning because the tissues are preserved by the freezing process and are kept in the preserved state while sectioning.
Kind Regards
Chris
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