>From: administrator_at_lrmc2__landstuhl@link.smtplink.lrmc.amedd.army.mil
>Date: Fri, 30 Jul 99 07:45:00 +0100
>To: <ccrowder@mail.vetmed.lsu.edu>
>Subject: cc:Mail Link to SMTP Undeliverable Message: Unknown user: ltc
margaret richardson
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>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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