Feulgen Reaction for DNA

1. Hydrolyze each set of standards and unknown slides simultaneously in 5 N HCl for 20-30 min at 21-23°C (see Note 6).

2. Dip the slides for 5-10 s in 0.01 N HCl (to keep the tissue at <pH 2 to keep aldehyde groups available and also to minimize the amount of 5 N HCl carried over by the slides that would drop the pH of the Schiff's reagent).

3. Put all of the slides of a set into 1% Schiff's reagent. Allow the tissues to stain at room temperature for 2 h. Faint pink staining will probably be evident after 10-15 min.

4. Rinse the slides in three 5-min changes of sulfite water (made up fresh each day). Keep the Coplin jars covered during each successive 5-min rinse to remove excess Schiff's reagent from the slides.

5. Rinse the slides in running tap water for 10 min. Color will intensify as the excess SO2 washes out of the tissue.

6. Rinse the slides in three 5-min changes of deionized water.

Decide which of the following steps (steps 7a and 8a, or steps 7b and 8b) is most appropriate for your purposes.

7a. For cytophotometry, dehydrate the slides through a graded series of ethanols to absolute alcohol and air-dry for storage in the dark until mounting in refractive index liquids for measuring. 8a. For cytophotometry, directly after staining, add three to four drops of an appropriate oil to clear the air-dried tissue by matching its refractive index (see Subheading 3.10.) and add a no. 1 thin cover slip to finish the preparation.

7b. To make permanent preparations, dehydrate the slides through graded ethanols to xylenes.

8b. Clear the slides from step 7b in three changes of xylenes, mount in a suitable plastic resin (e.g., D.P.X.). After adding a no. 1 cover slip, allow the preparation to dry overnight before viewing under an oil immersion lens.

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