Re: [Histonet] Melanin, fast red, and a counterstain

From:Gayle Callis


Red chromogens are excellent for giving a red/blue contrast with 
hematoxylin, and having the melanin pigment there too, you will have three 

Our researchers prefer red chromogens with hematoxylin, and we never use 
methyl green with red, but have used it with enhanced DAB (black 
color).  Most of our researchers are used to seeing hematoxylin stained 
sections and this counterstain is helpful for additional morphology 
examination.   Be sure to not be heavy handed with the hematoxylin, you 
want to LIGHTLY counterstain so you don't mask your red immunostained cells.

You can always counterstain one slide with hematoxylin and not counterstain 
an adjacent section just to see the different color contrasts, you may 
actually prefer NO counterstaining.

We never use methyl green with red chromogens, but have used it with brown 
DAB or an enhanced black DAB.

Be careful, methyl green may leach out of the section with a red AEC when 
you coverslip with aqueous mounting media as AEC will wash out/fade in 
alcohol or clearing agents.  Vector NOVA red is permanent and will not fade 
after exposure to solvents.   So be aware of mounting media compatibility 
with the chosen chromogen.

At 11:07 AM 10/30/2006, you wrote:
>Thanks to everyone who answered the questions about melanin pigment. It
>seems most of you use a red pigmented chromogen to demonstrate IHC
>staining that will be easy to differentiate from melanin. I should have
>asked this question at the same time: do the red chromogens work all right
>with a hematoxylin counterstain, with a methyl green counterstain, or can
>I do without the counterstain entirely? Thanks again.
>Melville B. Vaughan, Ph. D.
>Assistant Professor
>Department of Biology
>University of Central Oklahoma
>100 N. University Drive
>Edmond, OK 73034
>Histonet mailing list

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)

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