[Histonet] Methyl Green Counterstain; also the name

[John Kiernan]

Alcohol-water mixtures rather easily remove cationic dyes from stained tissues, and "methyl" green is particularly easily removed. Water alone also removes this dye, especially if it is slightly acidic. Ordinary lab distilled or deionized water is commonly at pH 5 because of dissolved carbon dioxide. This is only slightly less acidic than the "methyl green" staining solution (usually pH 4). NB Kurnick (Stain Technol. 30:213-230, 1955) overcame the problem by transferring the stained slides, without washing, to filter paper, blotting, and then placing in two 5-minute changes of n-butanol. After the second n-butanol the slides can be cleared in xylene and covered in the usual way. A very brief rinse in water (5 sec with agitation) is permissible before blotting the stained slides. n-Butanol is only partly miscible with water and it has an unpleasant, irritating smell. Some people use acetone instead (see J Brachet Quart J. Microsc. Sci. 94: 1-10, 1953). I prefer n-butanol.

We shouldn't really be calling the dye methyl green because that dye (CI 42585) has not been manufactured for perhaps 40 years. The dye sold under that name is really ethyl green (CI 42590), and it is greatly superior to the old methyl green. See Conn's Biological Stains, 9th or 10th ed, or The Sigma-Aldrich Handbook of Stains, Dyes and Indicators (FJ Green, 1990). Instead of mislabelling the bottles, vendors should be proudly selling this dye as ethyl green! 

John Kiernan
Anatomy, UWO
London, Canada
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----- Original Message -----
From: Igor Deyneko <igor.deyneko <@t> gmail.com>
Date: Friday, July 13, 2007 21:56
Subject: [Histonet] Methyl Green Counterstain
To: histonet <@t> lists.utsouthwestern.edu

> Hello.
> I am trying to do a Methyl Green counterstain on some cancer tissues,
> however the problem that I encounter is that the Methyl reen is 
> quicklywashed off the slides when done according to supllied protocol.
> Methyl Green is from DAKO.
> Protocol:
> 
> For optimal results, use the following procedure:
> 
> 1. Rehydrate tissue.
> 
> 2. Stain tissue sections for 5 minutes with Methyl Green.
> 
> 3. Rinse with 95% Ethanol to remove excess reagent.
> 
> 4. Dehydrate through graded alcohol.
> 
> 95% Ethanol 30 seconds with dipping
> 
> 95% Ethanol 30 seconds with dipping
> 
> 100% Ethanol 30 seconds with dipping
> 
> 100% Ethanol 30 seconds with dipping
> 
> Xylene or xylene substitute 2 minutes
> 
> Xylene or xylene substitute 2 minutes
> 
> 5. Mount in compatible permanent mounting media
> 
> 
> 
> Can anyone offer an alternative processing , maybe with 
> different times or
> alcohols?
> 
> Thank you.
> 
> Igor D.
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