We need to do double staining of FFPE using antibodies that require different HIER - one pH 6 and the other pH 9. Does anyone know if this is possible perhaps by doing HIER at both pHs in series before staining with the two primary antibodies together? I do know the epitopes are stable through several rounds of HIER at the same pH (6) followed by stripping/photobleaching and restaining (cyclic immunofluorescence). We likely will try different pHs but I wondered if anyone has any experience, knowledge or suggestions.
Thanks!
There is a theory, that antigen retrieval at a special pH followed by slow cooling will effect the folding of proteins in a specific way. This results in "presentation" of the antigen of interest.
So following this theory combining of high and low ph will result in the folding of the last AR-pH. Nevertheless masking of antigens by formaldehyd-adducts will be removed in a combined/doubled way.
It is likely, that both antigens are stained more intense. (morphology?)
Most antigens are stained better after high-pH-AR. So trying to use only high-pH-AR would be an option.
For chromogenic IHC you have the option to combine the IHC-rounds sequentially (DAB-FastRed).
Thanks for your reply. I am reminded of advice that rapid cooling immediately after HIER can give better results ("crisper" staining). See https://www.researchgate.net/post/How_do_you_choose_the_pH_for_your_antigen_retrieval_solution . Perhaps rapid cooling more reproducibly "captures" the epitope in the altered state that was induced by pH at temperature. The only solution is brute force trail and error but planning is still useful (I hope).
Ok. That's the opposite of what I have learned. After rapid cooling problems with section-adhesion and morphology may occur. Duration of heating and cooling should be always the same for standardization.
Probably it is the typical histotechnic-answer: "It depends ...."
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