On Tue, 4 Jul 2000, Kelvin Yen wrote:

> 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. ... 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.

If the EGP gives correct staining of DNA & RNA on similar sections,
your problem must be due to an effecto prior immunostaining. You do
not say what kind of sections you use, or how the immunostaining is 
done. If you provide more information, probably someone will come
up with an answer very quickly.

Among the things that could be messing  up your EGP counterstain
are:

  1. An antigen retrieval procedure might extract RNA, leaving only
     DNA to be stained by ethyl green and pyronine.
  2. If the estrogen receptors are in the nuclei of cells, deposition
     of antibodies and of enzymatic reaction products might interfere
     with the competitive binding of dyes by nucleic acids.
       How are you making the immunopositive sites visible? Peroxidase
     & DAB?  An alkaline phosphatase method? Fluorescence?  

Tell it all, and someone will tell you what you should be doing. 
It's pleasing to see that the true name of ethyl green is catching
on, after 25+ years!

 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1