Methyl/Ethyl green - pyronin + Immunostaining problem

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From:Kelvin Yen <>
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I've been trying to immunostain for estrogen receptors using an ABC
system and then following that up with a ethyl green-pyronin(EGP)
stain and have been having problems when I combine the EGP w/ the
immunostaining.  I was wondering if anyone has a protocol that I can
use that works for them or if anyone has an experience with combining
EGP and immunostaining.  My EGP staining protocol works just right by
itself and so does the immunostaining but the combination of the two
seems to cause the EGP stain to differentiate poorly and both dyes
stain the nucleus.
Any insights would be extremely appreciated.

-Kelvin Yen
Diamond Lab
University of California at Berkeley

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