IHC: frozen sections or paraffin-embedded tissues? - (Sep/12/2010 )

Hi,

I need to do some immunohistochemistry experiments but I don't know whether I should use frozen sections or paraffin-embedded tissues. The thing is that I am already used to working on frozen sections for IF experiments, but it seems that most people use paraffin-embedded tissues for IHC. Is there a particular reason for that? In addition, should I fix the tissues even when working with frozen sections?


Thank you very much.

bioche82 on Sun Sep 12 16:33:20 2010 said:

Hiya!

In our lab we routinely use paraffin-embedded samples and get good results.

I think a large part of the reason people choose to use paraffin samples is the ease of storage: samples can be stored at RT for long periods of time and still be successfully stained afterwards. Also, they can be easily cut using a microtome without the need for cooling.

Although there are reasons for selecting wax over frozen tissues, which are highlighted in the abstract of this paper.

As for your last question, I would say if your tissue/antigen of interest can handle being chemically fixed, then it's best to do so. It will help preserve morphology better than simply flash freezing.

Paraffin was traditionally performed due to processing and storing at room temperature, as Steffi said. However, if possible, I would recommend frozen section. Most antibodies do not work on paraffin sections, particularly anti-phospho antibodies. I know, if you perfom antigen retrievel etc pp, but I don't have that much time. Frozen section is also quicker to prepare, and the morphology is really well preserved. And I have looked at many different tissues, like hairy skin, whole bone, brain and testis. I never had problems with morphological changes. Background is also lower in frozen section (at least for confocal microscopy).
I would say: if you want to do H&E staining and look at morphology, use paraffin. If you want to do Confocal microscopy and look at the single-cell level or below, use frozen section (you can also do H&E with frozen section . So if you have a chance to do frozen section, do it. Best is, if you have enough time to compare both approaches. They give you very diferent results. And yes, you should fix the tissue when working with frozen sections.

I disagree that the morphology is better retained in frozen sections--In my experience morphology is much better retained in paraffin sections. However, I agree that the frozen sections are quicker--no dewaxing, rehydrating or antigen retrieval.