MGP Procedure

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From:rschoonh@sph.unc.edu (by way of histonet)
To:histonet@histosearch.com
Reply-To:
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>From one of my "old" files (18+ years ago).  I do not have the reference and
the notes are mine.   It has been a long time since I did this but I remember
how much fun the I had with the seperation (long story that I don't want to
get into).

                                METHYL GREEN-PYRONIN    STAIN  FOR  RNA AND
PLASMA   CELLS



                Procedure:




1. Use Carnoy's-fixed  paraffin sections if available.      Formalin fixed
tissue is adequate.
2. Run sections down to water.
3. Stain in MGP  reagent for 1/2  -   2  min.  (longer for old solution)

       4. Differentiate in 2 changes of distilled water.

      *5. Dehydrate  rapidly in 2 changes of acetone, clear in xylol, and
mount in Permount.

*Note:	If acetone removes too much of the red stain, slides may be blotted
dry with bibulous paper at this point.

Results:	Cytoplasmic and nucleolar ribonucleic acid (RNA): red.
Plasma cell
cytoplasm will also stain red.

            Note:	Always run a control section fresh fixed liver or
tonsil.



	Methyl Green-Pyronin Reagent

	Pyronine YÖÖÖÖÖÖ..0.25 gm

	   Methyl green. . . . . ÖÖÖ.0.75 gm

	   Phosphate buffer, pH 5.3Ö.100.0 ml

	To this add:

	0.5% phenol solutionÖÖÖÖ.0.5 ml

	1.0% resorcinol (fresh)ÖÖÖ..2.5 ml

Note 1:  The Methyl Green will need to be extracted with 3 changes of
chloroform in a separating funnel
Note 2:  The MGP reagent is relatively stable.  It will take 2-3 days to
ripen and will last 3-6 months if stored at  4 oC.

Phosphate Buffer Mixture

M/5 Disodium phosphate (Na2 HPO4 )ÖÖÖÖÖÖ 52.5 cc

M/10 Citric Acid  ÖÖÖÖÖÖÖÖÖÖÖÖÖÖ.. 47.5 cc

 Adjust to pH 5.3 (both of the above made up in 25% methyl alcohol to prevent
mold growth).

MGP for Smears

Use control slide.

1. Fix in 10% formalin methanol        10 min.

2.    Distilled  H2O 2 times.
3 .   Air dry 10-15 minutes.
4.    MGP reagent 5 minutes.
5..   Distilled  H2O 2 times
6.    Acetone 2 times.
7.    50/50 Acetone/Xylene
8.    Xylene
9.    Mount in Permount


best regards,
Bob
Robert Schoonhoven
Laboratory of Molecular Carcinogenesis and Mutagenesis
Dept. of Environmental Sciences and Engineering
University of North Carolina
CB#7400
Chapel Hill, NC 27599
Phone
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)

-- Begin original message --

> From: Louise Burrell <lburrell@pathbox.wustl.edu>
> Date: Mon, 22 Nov 1999 15:36:36 -0600 (CST)
> Subject: Re: Daily Digest
> To: HistoNet Server <histonet@pathology.swmed.edu>
>
> dear all-please URGENTLY email me with MGP stain for RNA and DNA---thanks
> ahead of time----i need it sooner than yesterday-
>
>                                 With warmest regards,
>                               Louise "Beezie" Burrell
>                               Chief Technologist-Cancer Center
>                               Tissue Procurement Core Facility
>                               Campus Box #8056
>                               Washington University School of Medicine
>                               St. Louis, MO.  63110
>                               Ph:  314-454-7615
>                               Fax: 314-454-5525
>
>
>

-- End original message --




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