Methyl Green/Pyronin

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From:Cheryl Crowder <ccrowder@mail.vetmed.lsu.edu>
To:HistoNet@pathology.swmed.edu
Reply-To:
Date:Thu, 29 Jul 1999 09:37:00 -0500
Content-Type:text/plain; charset="us-ascii"

I missed the first part of the MG/P discussion, so I'm not sure what has
been said.  However, I have several thoughts.  Methyl green manufactured in
the US is not very clean.  And it can take hours to clean it.  We have found
that MG from Chroma-Gesellschaft is very clen and can be used right away.  
        Our method for MG/P was given to us (years ago) by Pat Alexander
then of the Univ of Alabama Med School.  It does not use butanol or any of
the other nasty smelling organics but, rather, acetone.  I believe the
original article was in 'Histo-Logic".  It is very quick and easy:
        Sodium Phosphate, Dibasic   M15 (0.2M)
        Citric Acid M10 (0.1M0
        Phosphate buffer, pH 5.7 (McIlvaine's)
                Sodium phosphate dibasic, 0.2M      230 ml
                Citric Acid, 0.1M        170 ml
        Methyl Green-Pyronic, pH 5.3
                Methyl Green            3.0 gm
                Pyronin Y (or GS, not B)        1 gm
                Phosphate buffer                400 ml
                        Final pH should be 5.0 - 5.5

1.  Deparaffinize slides and hydrate to water.
2.  Stain in Methyl Green-Pyronin solution - 45 seconds to 3 minutes.
3.  Rinse slides quickly in distilled water.
4.  Blot slides dry using bibulous paper.
5.  Differentiate and dehydrate in 2 changes of absolute acetone (about 5
dips each).
6.  Rinse slides in 2 changes each of xylene-acetone (50:50).
7.  Clear slides in xylene and mount with Permount.

Results: 
        RNA - red
        DNA - blue

If the blue is too intense, tap water will remove it moe quickly than
distilled water.  If the red is too intense, 95% alcohol will remove it
effectively.  If both methyl green and pyronin stain too intensely, a weak
solution of acetic acid in 80% alcohol differentiates well.

There are several other "tricks" which can be used to help get great stains.
If you would like them, contact me directly.  Hope this helps.

Cheryl

Cheryl Crowder, Chief Technologist
Department of Veterinary Pathology
School of Veterinary Medicine
Louisiana State University
Baton Rouge, LA  70803
(225) 346-3399
FAX  (225) 346-3226





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