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FIXATION:        10% buffered neutral formalin.  Other fixative which may be used are Carnoy’s, formol saline and Zenker’s.

TECHNIQUE:        Paraffin sections cut at 5 μm.

SOLUTIONS:

0.2M Acetic Acid

Acetic acid, glacial --------------------------------------------------------------                3.0 ml

Distilled water -------------------------------------------------------------------        247.0 ml

0.2M Sodium Acetate

Sodium acetate anhydrous -----------------------------------------------------------        4.1 gm

Distilled water to make a total of ------------------------------------------------        -----  250.0 ml

0.2M Acetic Acid-Sodium Acetate, pH 4.2

0.2M acetic acid -----------------------------------------------------------------        75.0 ml

0.2M sodium acetate ------------------------------------------------------------        25.0 ml

       Check with pH meter and adjust to pH 4.2 with 0.2M acetic acid

       or 0.2M sodium acetate.  Store in a refrigerator at 3-6o C.

0.5% Methyl Green Solution

0.2M acetate buffer solution, pH 4.2 -----------------------------------------                10.00 ml

Methyl green, C.I. 42585 ------------------------------------------------------                0.05 gm

Methyl Green – Pyronin Y Solution

0.5% methyl green solution ----------------------------------------------------                10.000 ml

Pyronin Y, C.I. 45005 ----------------------------------------------------------                0.005 gm

       The amount of pyronin Y may need to be increased up to 0.01 gm

       with some samples of pyronin Y.

• Use control slides.

1. Deparaffinize and hydrate to distilled water.
2. Place on a staining rack.  Flood sections with methyl green – pyronin Y solution.  Let stand for 5 minutes.
3. Rinse quickly in distilled water.
4. Blot completely dry with blotting paper.
5. Place in acetone for 10 seconds.
6. Dip several times in equal parts acetone and xylene.
7. Clear in xylene, three or four changes.
8. Mount with synthetic resin.

DNA -----------------------------------------------------------------        blue to blue-green

RNA -----------------------------------------------------------------        pink to red

Background ---------------------------------------------------------        pale pink

This method produces better staining results than any other methyl green – pyronin Y method which we have tried on formalin-fixed tissues.

The dye content of pyronin Y will vary from sample to sample.  Usually the dye content falls between 50% and 90%.  This variation in dye content is what sometimes requires increasing the amount of pyronin Y in the methyl green – pyronin Y solution.

Some texts and staining manuals recommend that methyl green be chloroform extracted to remove methyl violet.  We have found that good staining results can be achieved with most samples of methyl green without chloroform extraction.

Potvin, C.:  A simple, modified methyl green – pyronin Y stain for DNA and RNA in formalin-fixed tissues.  Lab. Med. 10:772-774, 1979.