Exercise 2.5 - The Feulgen Procedure for DNA

LEVEL I

Materials

• Carnoy fixed, paraffin sections of tissue 2
• Oven at 60° C
• 1 N HCl
• Schiff Reagent
• Sulphurous acid
• Graded ethanol series, 50, 70, 95 and 100% (v/v)
• Xylol, Permount and coverslips
• Microscope

Procedure

1. Deparaffinize slides by passing through two changes of xylol. Rehydrate the slides by passing through 100%, 95%, 70% and 50% alcohols.

2. Rinse gently in distilled water (2 min.)

3. Pass the slides sequentially through the following:
   • Extract one slide in 1N HCl at 60° C (12 min.)
   • Extract second slide in 1N HCl at 60° C (1 hour)
   • Extract third slide in water at 60° C (1 hour)
   • Rinse gently in fresh distilled water (1 min.)
   • Stain in Schiff's reagent (30 min.)
   • Bleach with sulphurous acid (10 min.)
   • Rinse gently in fresh distilled water (1 min.)
   • 70% alcohol (5 min.)
   • 100% alcohol (5 min.)
   • Xylol (5 min.)

4. Mount in Permount.

5. Draw representative cells from each slide in the box provided. If Figure 2.4 has been completed, compare the cells stained with methyl green-pyronin to those stained with the feulgen reaction. Be sure to include a comparison of any enzyme or acid hydrolyzed controls.

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Drawing of feulgen stained cells Notes

The Feulgen procedure is undoubtedly the most widely used and most quantitative of all the cytochemical methods. Schiff's aldehyde reagent is the stain used. The great value of the Feulgen procedure is the manner in which cells are pretreated so that DNA furnishes the only available aldehyde to react with Schiff's reagent.

Extraction of cells with 1 N HCl at 60° C for 12 minutes provides optimum hydrolysis of purines from DNA, exposing the C1--aldehydes of deoxyribose.

The feulgen reaction is a semi--quantitative technique. If the only aldehydes remaining in the cell are those produced from the hydrolysis of DNA, then the technique is quantitative for DNA. It is possible to use an instrument known as a microdensitometer or microscpectrophotometer to actually measure the intensity of the pink feulgen reaction for a given organelle. Using this procedure, it was early determined that interphase cells were composed of two populations, those with 2C level of DNA and those with 4C. The nuclei looked identical, but one contained twice as much DNA within it.

This gave rise to the division of the interphase period of the cell cycle to a G1, an S, and a G2 based on the synthesis of that extra DNA.

For now, see if you can visually discern differences in the intensity of the feulgen stain reaction within the nuclei.

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