Exercise 2.4 - Methyl Green-Pyronin Staining of DNA

LEVEL I

Materials

• Cryostat
• Cold subbed slides
• Acid alcohol
• n-butanol
• 1 N HCl
• Methyl green
• Pyronin Y in acetone
• Incubator at 37° C
• Oven at 60° C
• 0.1% (w/v) RNAase in 0.1 M sodium phosphate buffer, pH 7.0
• 0.1 M Sodium phosphate buffer, pH 7.0
• Xylol, Permount and coverslips
• Microscope

Procedure

1. Remove a sample of tissue from a freshly sacrificed animal and freeze immediately for use in a cryostat.

2. Cut 7-10 sections and immediately fix in acid--alcohol (2 min.)

3. Rinse the fixed sections gently in distilled water (2 min.) and pass the sections through the following:
   
   • Digest one slide in buffered 0.2% RNase at 37° C (1 hour)
   • Digest second slide in 1N HCl at 60° C (1 hour)
   • Digest third slide in buffer only at 37° C (1 hour)
   • Rinse all slides gently in fresh distilled water
   • Stain in 0.2% methyl green (4 min.)
   • Blot excess methyl green from slides
   • Rinse in n-butanol (5 min)
   • Stain in 0.6% pyronin Y in acetone (1 min.)
   • Xylol (5 min.)

4. Mount in Permount

5. Observe and draw the tissues in the space provided. Compare the cells treated with RNAase to those treated with HCl and buffer only.

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Drawing of methyl green - pyronin stained cells

Notes

The methyl green-pyronin procedure uses the high net negative charge of nucleic acids. Methyl green is a cation which binds rather specifically to DNA and thus serves as a convenient means of staining nuclei in both f ixed material and living cells. Pyronin, a red dye, is fairly specific for RNA with some binding to protein.

Control slides are important in interpreting the results of methyl green-pyronin staining, since the procedure is readily susceptible to artifact. One or both of the nucleic acids should be removed either enzymatically or by acid extraction.

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