[Histonet] RE: Methyl Green
The brown-red color combination you mentioned allows a simple
hematoxilin counterstain. I would advise you to work with a diluted
hematoxilin solution and keep the time short. Check out the
counterstaining result microscopically and if needed repeat.You don't
want intensely blue nuclei as this will mask your double staining.
If you really wish to counterstain with methylgreen soak your slides
in 100 mM Na-acetate buffer pH 5.2 for 15 min. Don't wash. Cover
sections for 5 min with 0.1% methylgreen (Sigma M6776) dissolved in
the acetate buffer as above. Wash briefly with with distilled water
and dry the slides completely at a hot plate. Mount organically with
VectaMount. This procedure circumvents dehydration by alcohols, which
isn't good for your red alk. phos. reaction product.
Chris van der Loos, PhD
Dept. of Pathology
Academic Medical Center M2-230
NL-1105 AZ Amsterdam
phone: +31 20 5665631
fax: +31 20 6960389
Date: Tue, 27 Sep 2005 14:19:05 -0700
From: "Eeva, Mervi"
Subject: [Histonet] Methyl Green
I tried Methyl Green counterstaining on my double stained
immunohistochemistry slides and didn't get any visible
Tissue was FFPE brain and otherwise I used mostly Vector reagents with
and AP-red detection.
Any ideas what went wrong?
- Mervi Eeva, UCLA
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