Re: wrong secondary

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From:Amos Brooks <>
To:Joyce Kotzuk <>
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    I wonder ... If acid will remove the primary antibody from the tissue, would an acid rinse in the counterstaining steps affect the signal intensity?
    As for the wrong secondary, I have recut slides and performed IHC staining side by side with rerunning the same IHC procedure starting from the proper secondary. (Dont start from the H2O2 or the tissue will be overquenched, and will probably fall off.) They both worked OK. The only problem I had was a slight background on the first set. It was a small price to pay since the specimens were tiny prostate biopsies, and therefore after being cut, recut, then cut again, much of the specimen was lost. Rule of thumb for this ... Beggars can't be choosers!
    I also noticed the earlier you catch the error the better. If the slide is already coverslipped, there will probably be more problems than if you catch it right after the application of the wrong secondary.
Amos Brooks

Joyce Kotzuk wrote:

> There was some discussion here recently about eluting primary antibodies off of tissue by using an acid, something below pH 2.5-3. If you could get the primary off, the wrong secondary may come off with it, and then you could start over from the beginning.  Otherwise, if your primary was used at a concentration which was determined to be optimal for getting good signal, I don't think putting more primary on would result in any additional staining.  I would be interested in hearing again about the acid elution of primary antibodies anyway, specifically, if the primary comes off, DOES the secondary, ABC and DAB also come off, or just the secondary and ABC, leaving the precipitated DAB in place?
> Joyce Kotzuk
> Univ. of New Mexico neuropathology lab

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