Exercise 2.4 - Methyl Green-Pyronin Staining of DNA

LEVEL I

Materials

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:

  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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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- cellab@gac.edu