I want to first optimize two antibodies and then move on to double staining. I feel like it might be a good idea to optimize them using a general protocol, but later on to save time, it might be feasible to use a microwave if possible.
The tissues that I will be sectioning and staining were frozen and embedded in OCT at -80, but are not embedded in paraffin.
I know that antigen retrieval of paraffin-embedded tissues can be done using a microwave, and that microwave can help with rapid fixation of frozen sections. But is it also useful for the subsequent staining steps (for frozen sections NOT embedded in paraffin)?
One of the suitable materials for IHC is frozen sections due to the absence of the need for epitope retrieval and perceived preservation of antigenic sites. In view of that you may go ahead to optimize your antibodies and carry out the double staining without necessarily using the microwave. Just follow the manufacturer's manual for the incubation time, in which the optimal temperature may also be stated depending on the method (for instance with fluorescent IHC the recommended incubation temperature is 2- 8 oC overnight).
Hi Gargi,
Antigen retrieval is often useful for improving immunolabeling in frozen sections. Our experience is that antigen retrieval is essential to achieve staining for some antigens in our frozen section preparations (mostly retina and brain). Whether antigen retrieval is needed depends on the target antigen and the antibody to be used. There are a number of antigen retrieval approaches in use. For our own work, we have found that hot citrate buffer is pretty effective, but some antigens may require different approaches. I've attached a copy of our standard frozen section immunolfluorescence protocol in case its of use to you. Good luck.
Dave Sherry
In my experience I have never had to use antigen retrieval in frozen sections since there is no paraffin to block antigen sites.
I don't recommend using a microwave oven for any histologic technique. It has been proven to "cook" the tissue since the heat within the oven is not consistent in every area of the interior.
Dear all,
thank you so much for these insights!
I will post more questions as they come this year.
Best,
Gargi
Hi Gargi,
I try to avoid antigen retrieval if possible, as it tends to result in a higher background or nonspecific labeling. As a result, I prefer to use frozen rather than FFPE samples, as the later always requires antigen retrieval but frozen tissues do not.
If you do try antigen retrieval, try both citrate (pH 6.0) and TE (ph 8.0-9.0) buffers. Microwaves can work, but I prefer a constant source of heat, such as a water bath. I have also heard of people using pressure cookers or automated rice cookers for antigen retrieval.
Good luck!
I did pressure cooker, microwave, water bath and steamer for antigen retrieval. For frozen tissue you should do gentler cook, I use steamer with a thermometer probe to stick in the retrieval solution to monitor the temperature. I use pressure cooker for paraffin tissue if tissue stays on the slide.
Until you evaluate the quality of staining with no treatment (such as epitope retrieval) you may find that all is good enough to proceed to double labeling. You should look at the Shindler and Roth 1996 paper that emphasizes the use of amplified fluorescence methodology along with conventional direct-tagged secondary approaches. It is powerful and highly useful. In evaluating staining, I always would use amplification methods first since they are about 100x more sensitive. Here is a how-to paper for freely floating sections. It works well for paraffin and frozen sections as well but frozen sections of unfixed tissue can impair penetration of antibodies. That said, we have used it successfully
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