I am trying to do antigen retrieval on mouse brain sections to stain for proliferative markers like MCM2, Ki67, PCNA etc on tissue that has mCherry conjugated to a transcription factor of interest to out lab. However, most AR techniques seem to kill the mCherry signal after staining. I have use many different heat induced AR methods with a variety of citrate buffers but any antigen retrieval that brings the tissue above boiling seems to kill the signal. What are some other options I may want to pursue?
Hi William Meara,
I would like to suggest you some strategies that might be useful in order to try to overcome these difficulties you are experiencing. First of all and without considering modifying your tissue processing protocol, have you tried using an antibody against mCherry itself so that, despite having lost its intrinsic fluorescence due to sample heating, you can still detect its presence? I believe this could work but, if not (or if you have already tried), you could also carry out enzymatic-based (commonly trypsin) antigen retrieval procedures. Although I am not very familiar with them, they surely do not require severe heating - just the optimal temperature for the reaction to take place (i.e. 37ºC), and may preserve mCherry fluorescence to a higher level. On the other hand, if your tissue processing method includes paraffin infiltration, you might want to consider performing agarose embedding and subsequent vibratome slicing instead. Although it is true that the thickness of the sections you can obtain is a little higher in comparison with the paraffin-based method, I strongly recommend it since it is also a really less aggressive treatment for many epitopes as sample heating is not needed. And, in such case, you may be able to avoid the use of antigen retrieval - in our hands, proliferation markers (except BrdU) do not require it in mouse brain tissue. If you are already working with vibratome slices, then the two first options (specially the use of a mCherry antibody) could be worth taking into account.
I really hope these suggestions might be effective and could help you conduct your experiments!
Kind regards,
David
In support of David's response you might check out this article:
Article Immunohistochemical Amplification of mCherry Fusion Protein ...
Other forms of enzyme based antigen retrieval that you could try include: proteinase K, triton-x 100, tween 20, etc. However, if you are required to use HIER for the other antibodies, then it seems that detecting mCherry by IHC would be your best bet.
Good luck!
Melissa
Hi Meara,
I think the easiest way is to use antibody to label endogenous reporter, like mCherry. I used GFP antibody to label endogenous reporter in paraffin section during immunofluorescence, and the staining is good and specific. Hope it will help you.
Best,
Chenglei
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