- Fixation can alter protein biochemistry such that the epitope of
interest becomes masked and is no longer assessable by the
probe. Citrate boiling is one technique of antigen retrieval in
which the masking of an epitope is reversed and epitope–probe
binding is restored. - This treatment will inactive endogenous phosphatase activity
and reduce background staining. - It is important to maintain the temperature above 100Cto
ensure successful antigen retrieval. - ACD sells a hybridization oven that addresses the aforemen-
tioned concerns very well. However, I find that using a mod-
ified Tupperware and a standard incubator works equally as
well. - I find that using a P1000 pipette tip to spread the solution is
helpful to achieve complete coverage. - We have not noticed changes to signal intensity or background
noise with longer incubation times - At this point, you can counterstain the slides with 50% hema-
toxylin solution using standard staining protocols. However, in
our experience, counterstains have a tendency to dampen sig-
nal intensity.
Acknowledgment
This work was supported by Institutional Development Awards
(IDeA) from the National Institute of General Medical Sciences
of the National Institutes of Health under grant numbers P20-
GM104318 and P20-GM103423, and an American Heart Associ-
ation Scientist Development Grant (11SDG7210045) to V.P.Y.References
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