I would like to know if it is possible and if yes how to store cryosections which were fixed with 4% PFA after sectioning? I only find protocols which immediately go on with the staining procedure after the fixation so I am asking myself is this somehow a "no-go" or is there a specific reason why the post-fixation is always done right before the staining? I really hope someone can help me with this question. Thank you!!
If these are sections after fixing the sample, infusing with glycerol or OCT and then sectioning, at -20 or so, you can store the sections in a -80 freezer for a fair amount of time.
Are you talking about TEM or light microscopy sections? After cryo-sectioning, I assume you are warming the sections to room temperature for fixation. For TEM samples, you can invert the grids and float them onto PBS in a small petri dish and place them into the fridge after the initial PFA fixation. I have done this from overnight to a couple of days before further processing (immunolabelling in my instance). I've not done this for light microscopy sections but think storage completely covered by PBS in a covered petri dish in the fridge would also work. You cannot store your material too long in just PBS as fixation by PFA is reversible (very slow process though).
Thanks for the answer:) So I am talking about light microscopy sampels in my case. To make my point a litte bit clearer I will just shortly describe my working procedure. So I collected intestinal tissue and without fixation I embed it directly into OCT compount and the sampels are stored at -80°C. After I collected all my samples, I sectioned the samples in a cryotome (5 µm) and let them dry. After that I fixed the samples with 4% PFA and then again let them dry and stored them in the fridge. What I am not sure about is if there is a problem with the drying after the fixation and if yes, whats the problem about it? Maybe I missed some really basic principle but I really don't get why this should be a problem.I hope that makes my question a little bit clearer
Our lab tried a 10s fixation with 100% methanol immediately after sectioning. Like your case, we don't fix the tissue before we freeze the tissue in OCT at -80C. The morphology looks fine.
To let tissue dry never is a good way of handling. It would have been better to let the tissue in neutral buffered formalin and store the container in the fridge. It depends on your following aims if storing in the fridge is really necessary.
Drying causes tissue shrinkage, sintering together of tissue structure. It is comparable to processing the tissue from formalin directly into absolute ethanol for dehydration. There is a reason, why we do stepwise tissue processing over graded ethanols.
This protocol seems very rough on the the sample, potentially several areas of artifact may be inserted. Of primary concern is the lack of cryoprotection prior to freezing of the sample at -80C. Secondly, why are you not immersion fixing prior to freezing if you plan on using a fixative later? Finally, what are you staining for/how? Simple histology like Cresyl Violet is forgiving, Fluorescence not so much.
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