Re: Methyl green

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From:Robert Schoonhoven <>
To:Lynn Gardner <>
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My procedure is as follows, I have sent this one out a time or two and
no one has complained about it.  It works very nicely in my lab..

Methyl Green Counterstain

a)	Acetic Acid, Glacial (aldehyde-free) 
b)	Sodium Acetate, Anhydrous 
c)	Methyl Green


1 )    0.1 N Acetic Acid: Add 6 ml of glacial acetic acid to 1000 ml of
filtered distilled water.  

2)	0.1 N sodium Acetate: Add 4.102 g of sodium acetate to 500 ml of 
distilled water. 
  3)      Buffer:
   a) Add 755 ml of 0.1 N acetic acid to 265 ml of 0.1 N sodium acetate.
   b) Adjust pH to 4.2 with NAOH.

4)	Methyl Green 
a)	Add 20.0 g of methyl green to the buffer solution.
b)	Filter the methyl green solution.

STORAGE: room temperature
SHELF LIFE: Indefinite while in storage; when in use, the shelf life is
2 weeks or until staining becomes too weak.

1)	After color development of slides, rinse in distilled water briefly.

2)	Place slides in methyl green solution for 3 to 5 minutes, or as long
as necessary to get the
         staining desired. (slides which have been denatured, i.e. as
for in-situ-hybridization, 
         typically need far less Counterstain 30 seconds or so.)

3)	Do not rinse in water.  The methyl green washes out too easily.  Go
directly into 95%  
          alcohol for a brief time (about 10 dips) .

4)	Continue dehydrating through one more 9S% alcohol, then 3 changes of
100% alcohol,   
         allowing the slides to stay in the last alcohol for 2-3

5)	     Clear through 3-4 changes of xylene, and coverslip.

Lynn Gardner wrote:
> Hey all, I know people have been discussing this lately however, I didn't
> pay close enough attention.
> I am in need of a methyl green counterstain for alkaline phosphotase
> stained tissue that will be crisp and consistant and won't decrease in
> intensity over time. Does anyone know of such a critter???
> Thanks all
> Lynn Gardner

best regards,
Robert Schoonhoven
Laboratory of Molecular Carcinogenesis and Mutagenesis
Dept. of Environmental Sciences and Engineering
University of North Carolina
Chapel Hill, NC 27599
office 919-966-6343
   Lab 919-966-6140
   Fax 919-966-6123 

Don't go around saying the world owes you a living; the world owes you
nothing; it was here first. 
Mark Twain [Samuel Langhornne Clemens] (1835-1910)

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