Re: methyl green as counterstain

From:Robert Schoonhoven <>


The following has worked very well for me over the years.

Methyl Green Counterstaining  Procedure


0.1 N Acetic Acid

0.1 N Sodium Acetate

Methyl Green


Add 755 ml of 0.1 N acetic acid to 264 ml of sodium acetate and adjust 
the pH to 4.2 with NAOH

To the above add 20 gm Methyl Green.

Can be stored at room temp.  

If staining turns bluish than the solution has started to deteriorate.


1- Rinse in distilled water briefly
2- Place slides in methyl green for 3-5 minutes (or longer ) 
3- Do NOT rinse with water.  Go directly into 95 % EtoH for about 10
dips, then continue through 1 more 95% and 3 100% EtoH changes leaving
the slides in the last absolute for 3 - 5 minutes.
4- Clear with 2-3 changes of xylene and coverslip

Tina Cardamone wrote:
> Hello,
> I have a simple question regarding methyl green.
> Does anyone have a methyl green counterstain  protocol for
> immunohistochemistry.
> It sounds simple however I can't seem to find a protocol anywhere.
> Thanks in advance.
> Tina Cardamone
> University of Melbourne.

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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