RE: [Histonet] Water stable counterstain?

From:"Smith, Allen"

   There are two silver stains for nuclei that are completely
water-resistant:  Korson's 1964 (J. Histochem. Cytochem. 12: 875)
methanamine-silver method for DNA and Black and Ansley's 1964 (Science 143:
693) ammoniacal silver method for histones.  It has been 35 years since I
used either method, but my recollection is that Korson's methanamine-silver
is extremely fussy and time-consuming.  Black and Ansley's ammoniacal silver
is quite tricky at first, but fairly simple once you get the hang of it.
   Mayer's paracarmine (p. 84 of the 2nd edition or p. 215 of the 4th
edition of Lillie's HISTOPATHOLOGIC TECHNIC) calls for 70% alcohol for the
staining solution, but the resulting stain is water-resistant.  I used to
make up a stock solution in 70% alcohol and dilute it to 50% alcohol for
staining.  Other alum carmine methods would probably also give
water-resistant staining.

Allen A. Smith, Ph.D.
Professor of Anatomy
Barry University School of Graduate Medical Sciences
    Podiatric Medicine and Surgery
Miami Shores, Florida  33161

-----Original Message-----
[] On Behalf Of I.B.
Sent: Friday, September 29, 2006 7:23 PM
Subject: [Histonet] Water stable counterstain?

Hi All,

I just sent a long message about ISH, but I have another question perhaps
someone can answer.  I am using Nuclear Fast Red (Vector Labs) to counter
stain tissue stained with NBT/BCIP (also Vector Labs), and, in order to
reduce the number of steps in the protocol, I would like to use an aqueous
based mounting media (at least with the aqueous media I have tried).
However, Nuclear Fast Red washes out of the tissue when mounted aqueously,
making contrast under the microscope terrible.  Can anyone

1) recommend a counterstain, preferably something red, that will be stable
in aqueous mounting media, i.e. not wash out?
2) recommend a water-stable non-red counterstain that has excellent contrast
to NBT/BCIP ( i.e., is there a different color counterstain that is
compatible with NBT AND aqueous mounting media?)
3) recommend a completely different substrate/counterstain combination that
works with DIG/Alkaline Phosphatase and that can be mounted aqueously?  (
i.e., if the NBT/BCIP just plain sucks, and I am getting the impression that
it does, is there anything out there I can use with Alkaline Phosphatase so
I don't have to completely rework my probes?)

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