Tissue preparation for confocal microscopy: frozen vs paraffin.

It was shown that freezed sections are not worse than paraffin-embedded for immunohistochemistry, if made accurately, but I have no exact info on phosphoproteins. If you are not very skilled in preparing freezed ones, use paraffin.

I agree. Tissue morphology will be better with paraffin-embedded tissues (less stretching/tearing of the sections), but antigen retrieval & recognition are greatly reduced for many antigens. If you have a low-frequency antigen (dim on flow cytometry) or one that is particularly fragile, frozen sections (OCT) work better--but the tissue morphology gets distorted more easily. It was previously recommended that Zamboni's fixative could improve antigen detection in paraffin-embedded sections, but I never got to try it personally.

From my experience, frozen sections are better when looking at sensitive proteins. Paraffin will allow you to observe morphology/pathology a lot better on a "global" tissue scale, but determining the proper antigen retrieval becomes challenging (especially phospho-proteins).

Frozen sections work well for phosphoproteomics.Tissues or sections needs to be fixed with 4% paraformaldehyde.then then follow the regular protocol.

I suggest it is possible to use every kind of fixation for confocal microscopy. However, please, check the availability of antibodies for your target antigenes whether they works on frozen or paraffin sections. For example, you may use this link to do it: www.biocompare.com. Follow the staining protocol for your selected antibodies. On paraffin sections it may require an additional procedure of epitope retrieval (press-cooker treatment for example).

I believe paraffin sections will be easier to cut and might provide a better final tissue preservation when compared against OCT-embedded cryosections. On the other side, it envolves more critical steps that might both degrade antigenicity and will take significantly more time to obtain results. On my personal experience, I have been using aldehyde fixation, sucrose cryoprotection and freezing at OCT (Tissue-Tek) media as a method of choice if I already know my tissue morpholgy well-enough and need to maximize antibodies signal. OCT microtomy is a lot more tricky though.

I have done confocal with paraffin and had no issues. Honestly paraffin sections are much easier to work with during the staining/handling procedures than frozen. Depending on the tissues, frozen sections are extremely hard to cut and attach to slides, and if you have a histology core sometimes they will not do frozen, meaning you would have to cut them yourself (a nightmare, trust me). Frozen sections also often don't look as nice as paraffin. Frozen sections are ideal for doing enzyme related staining (things like succinate dehydrogenase staining), however for what you are talking about I would go with paraffin.

If you have frozen specimens, would you not have to keep them frozen during imaging? I would think you would first have to have some way to keep the samples from degrading if frozen. In my experience, paraffin has always been the choice for confocal work. Antibody labeling is always questionable in terms of binding and specificity (i.e. how good is the antibody against the selected compound/tissue). Regarding the above response, some protocols differ in terms of labeling before or after fixation. I typically label and incubate post-fixation for confocal depending on tissue thickness. Hope this helps.

hi carmen,
1. if your looking for more depth within tissue my vote will be for multiphoton, confocal cannot access great depths as MP, however with within sizing limit you can always switch between confocal or MP.
2. how are you planning to antibody label the specimen! my guess would be labeling them in live tissues before processing!
3. if your planning after fixing the tissue samples, i am sure it would be a great trouble getting your antibodies penetrated to the sites.
So safe to say i would prefer labeling them before embedded in paraffin
cheers