[Histonet] RE: Alkaline Phospahtase and Methyl Green

C.M. van der Loos c.m.vanderloos <@t> amc.uva.nl
Tue Mar 25 03:01:04 CDT 2008

Igor,The fact that Vulcan Red provides a good result whereas DAB showed strong background might be explained by the lower staining sensitivity/efficiency of the Vulcan Red compared with DAB. Have you tried to further dilute your primary antibody when using DAB?Using Vulcan Red as chromogen counterstained with hematoxylin usually gives an excellent result. Take care that the hematoxylin counterstain is not too strong and overwhelming the red reaction product. To prevent overstaining with hematoxylin dilute the standard Mayer's hematoxylin 1:10 with tap water and stain for 3-5 min. If you want to go for methylgreen, purchase Sigma M6776 (which is basically 'ethylgreen') and prepare a 0.1% solution in a sodium acetate buffer (0.1 M, pH5.2). First, incubate your slides with the sodium acetate buffer for 10-15 min, blott off and stain with the ethylgreen solution for 5 min. Wash with tap water (5 min) and dry slides at a hot plate. Coverslip with an organic mounting medium without xylene or alcohol (for example: VectaMount, cat.no. H-5000)Lots of success!Chris van der Loos, PhD
Dept. of Pathology
Academic Medical Center M2-230
Meibergdreef 9
NL-1105 AZ Amsterdam
The Netherlands Date: Mon, 24 Mar 2008 11:31:48 -0400
From: "Igor Deyneko" <igor.deyneko <@t> gmail.com>
Subject: [Histonet] Alkaline Phospahtase and Methyl Green
To: Histonet <@t> lists.utsouthwestern.edu

Dear Histonetters!
I was wondering if anyone can help me out. I'm doing IHC with Ach antibodies
and when I use DAB chromogen I get strong background. I used Vulcan Red
(from Biocare Medical), which got rid of the background, but it's a bit hard
to distinguish from Hematoxylin counterstain that I use. I was wondering if
Methyl Green would be an appropriate counterstain for it? I tried
using 0.5%Methyl Green from Plyscientific before, but had problems
because it washed
off right away in the first change of 95 Ethanol. I also tried n-butanol and
acetone, but the results were unsatisfactory. If anyone knows good
percentage to use as a counterstain and a protocol would be greatly
Thank you in advance.
Igor Deyneko
Infinity Pharmaceuticals
In-Vivo Pharmacology
Cambridge, MA

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