Hi. I am doing an immunofluorescence on paraffin sections using 5hmC and 5mC antibodies. I have managed to get them to work after antigen retrieval and incubation in 4N HCl, however my DAPI didn't work. I know for sure that my DAPI is fine because it worked on the slides that I didn't treat with acid, but only did an antigen retrieval on. Does anyone have any ideas of why it didn't work and how I can get DAPI stain on my sections after this treatment? Thanks.
The acid treatment most likely destroyed your DNA to an extent that DAPI cannot bind to it anymore well enough to produce significant fluorescence. DAPI seems to intercalate at AT-rich regions. If the DNA is too damaged (e.g. after acid treatment) then this may not happen anymore. Furthermore, DAPI seems to work best on intact RNA at a pH around 6 (http://onlinelibrary.wiley.com/doi/10.1002/1097-0320(20010101)43:1%3C12::AID-CYTO1013%3E3.0.CO;2-N/full). Even if you did not destroy most of the DNA with the acid, you may prevent DAPI fluorescence in low pH.
Why did you treat with acid? 5mC stainings should work on FFPE tissues without any acid pretreatment just with heat pretreatment. Do you use FFPE tissue sections? I have used microwave and pressure cooker pretreated FFPE tissue sections with a 5mC antibody (was sold by Santa Cruz Biotech, but they discontinued it; but the same clone can still be purchased from e.g. Active Motif or abcam).
To answer your question: I don't think you can resurrect the DAPI stain after harsh acid treatment.
Why do you treat your slides with acid? If your antibody requires slight acidity, I would change the pH of the antigene-retreavel buffer that is used but would not go bellow pH6-5.
I tried using just antigen retrieval in TE buffer, but unfortunately that didn't work. A lot of protocols suggest doing an acid step after antigen retrieval. It worked, there is binding of the antibody after doing the acid step, but unfortunately I can't get DAPI to work. I've seen it done in cell culture, but not sure how to get this to work on paraffin sections.
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