RE: Methyl Green pyronin staining

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From:"Kellar, Eric" <kellarec@MSX.UPMC.EDU>
To:"' '" <>
Date:Wed, 28 Jul 1999 16:30:32 -0400

Fixation does effect the DNA and RNA staining through denaturation and
depolymerization prior to MGP staining. I see lymph node biopsies stained
daily with my own prepared MGP after NBF, Zenker's and B5 fixation. The
pyronin binding to the RNA seems enhanced while methyl green's affinity is
markedly decreased from exposure to B5 fixation at 2-4 hours. I have omitted
the butanol step (used in many MGP variations) and replaced it with a cold
80% ethanol followed by absolute acetone to decolorize the tissue after
staining 10 - 15 minutes with brilliant and consistent results. The key, as
others have pointed out, is the meticulous preparation of the working methyl
green-pyronin Y solution. 

Eric C. Kellar
University of Pittsburgh Medical Center

From: 	Sarah Christo[]
Sent: 	Wednesday, July 28, 1999 9:09 AM
Subject: 	Re: Methyl Green pyronin staining

Dear Russ,
    How important is the fixation in the MGP stain?  I have one researcher
from Egypt here requesting the stain, but his tissue is all fixed in NBF.
Also, how important is the tertiary butanol?  Can n-butanol be used instead?
I found some very interesting comments in "Animal Tissue Techniques" by
Gretchen Humason, 2nd Ed. page 277.  She sited a MGP stain procedure by
Kurnick, 1952 were he used n-butanol to differentiate the methyl green, left
the pyronin out of the first stain solution and made the pyronin a separate
step after the methyl green.  The pyronin was saturated in acetone.  Any
thoughts on this?
  What is the title of John's book you sited?
Many thanks,

Sarah Christo, HT (ASCP)
Texas A&M University
College of Veterinary Medicine
Dept. of Vet. Anatomy & Public Health
College Station, TX  77868-4458

>>> RUSS ALLISON <> 07/28 7:51 AM >>>
The comments on "cleaning" methyl green are wellvery well made.

One comment to add - it IS possible to purchase pure methyl green.  
Merck sell it and the BSC certify it.  Hans Lyon did find that it was 
not always pure or even methyl green.  What he found on examination 
was that it could sometimes be, or be contaminated by, ethyl green.
Importantly, it made no difference to performance (in MGP 
staining), whether it was methyl or ethyl.

As has been so well described in earlier correspondence, it is the 
oxidation product that causes the problem.

I think it is Barka & Anderson's book - Histochemistry -  that 
describes the use of tertiary n-butanol  for dehydration after 
staining and which helps keep the green staining green, not blue.

See also the latest edition of John Kiernan's most excellent book
ISB 0 7506 3106 6

Russ Allison, Wales

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