Re: Need replies today!!! (counterstain for cultures)

From:"J. A. Kiernan" <>

On Fri, 30 Mar 2001, Greg Dobbin wrote:

> I have a graduate student doing ISH on cell cultures. The cultures 
> are grown on PLASTIC culture slides. He wants to counterstain with 
> methyl green (has his heart set on it, actually!!), but because of the 
> plastic, he cannot use acetone or xylene on his slides. Is there a 
> solution that will allow him to continue with methyl green? Any 
> suggestions for alternatives? Neutral red? Method? His substrate is 
> blue. Thanks. Greg
If he wants a pure nuclear counterstain the best bet
is probably an alum-brazilin or a carmalum. Make brazalum 
the same way as Mayer's haemalum, but with brazilin instead 
of haematoxylin. The staining solution is more stable than
the haemalum - keeps for 5 years+. For Mayer's carmalum,
you need carminic acid and an old recipe. This solution
does not keep so well. Another possibility is aluminium-
nuclear fast red. Experience with this for counterstaining
cultures in our labs has been rather disappointing. 

Although there's uncertainty about how these red dye-mordant
mixtures stain nuclei (just as there is with haemalum),
there is no doubt that they are much less easily extracted
into an aqueous mountant than a basic dye like methyl green
or neutral red, and of course the basic dyes aren't selective
for nuclei either. I can't think of a green nuclear stain that's
equivalent to haemalum. Probably someone else will come up
with one!
John A. Kiernan
Department of Anatomy & Cell Biology
The University of Western Ontario
London,  Canada   N6A 5C1

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