[Histonet] Re: counterstain for ABC-HRP (Neurons)

[John Kiernan]

If the HRP is detected with regular DAB, 
giving a brown product, you can use a blue
cationic dye to counterstain neurons. 

A thiazine dye is easier to use than "methyl
green." (That dye that has been deliberately 
sold under a wrong name for 30+ years; it's 
really ethyl green, which is much better; 
old timers can tell you why - but I'm getting 
off-topic). Toluidine blue is my favourite
thiazine; use it at 0.5%, pH 3 to 4. Azure A 
is just as good. So, probably, are the other 
azures and thionine, but I've not used them 
myself for this purpose.

>From your summary of the research, I assume
that you are working with monolayers of 
cultured cells. If the cells are on plastic,
you probably won't be able to use a resinous
mounting medium. (Its solvent is likely to
dissolve the plastic.) Aqueous mounting
media generally extract basic dyes ("bleeding"),
so you will need to use a trick to render the
blue dye insoluble in water. It's an old one
that dates back to Paul Ehrlich in the 1880s.

When the counterstain looks right under a
X10 objective, in a preparation that has been
rinsed in distilled water after staining, pour
on a 5% aqueous solution of ammonium molybdate
and leave it there for 5 minutes. Wash in water
and mount. The molybdate complexes (salts?) of 
the thiazine dyes (methylene blue, toluidine
blue, azures etc) are insoluble in water, acids,
alcohols etc. I have used this strategy to do 
Van Gieson (pH 1) after Nissl staining with 
toluidine blue at pH 4. 

If the neurons are not stained by your 
immunohistochemical method, they will have
strongly blue cytoplasm. Cells other than 
neurons will, for the most part, have only
their nuclei blue. If you have cells with
high rRNA content that are not neurons, they
will have blue cytoplasm, but their shapes
will be very different.  

If your neurons are immunopositive, they will 
be blue+brown - not a lovely combination. 
You asked for a counterstain, so I assume 
that the immunostain is for cells other than 
neurons.
-- 
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John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
   kiernan <@t> uwo.ca
   http://publish.uwo.ca/~jkiernan/
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"Atoska S. Gentry" wrote:
> 
> Hello,  Is anyone using a counterstain other than methyl green that stains
> neurons induced from bone marrow stem cells with the Vectastain ABC-HRP
> system? Thanks, Atoska
> 
> Atoska S. Gentry B.S., HT(ASCP)
> Research Assistant III
> Scott-Ritchey Research Center
> College of Veterinary Medicine
> Auburn University, AL  36849
> Phone# (334)844-5579  Fax# (334)844-5850
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