FEULGEN STAINING PROTOCOL
The Feulgen technique selectively stains DNA, and under controlled conditions, can be used for the photometric determination of DNA content. The reaction consits of two steps. Fixed material is treated for 8-10 min with 1N HCl in a water bath or oven at 60°C. Afterwards, the material is immediately transferred into Schiff's reagent at room temperature (for at least 30 min or until the tissue stains deep purple). The material is then squashed in acetocarmine or aceto-orcein. It is recommended the the material be analyzed the same day, however, it can be kept at 4°C for a several days if necessary.
Acid hydrolysis removes purin bases from the DNA, thereby unmasking free aldehyde groups. The aldehyde groups then react with Schiff's reagent, which results in the purple staining. RNA is not hydrolyzed by the HCl treatment and, thus, the reaction is DNA-specific.
Schiff's reagent
Schiff's reagent is prepared by pouring 200 mL of boiling distilled water over 1-g basic fuchsin. Shake thoroughly, cool to 50°C, filter, and add 30 mL 1N HCl to the filtrate. Cool to room temperature and add 1 g potassium metabisulfite (K2S2O5). Allow the solution to stand overnight in the dark or until a light straw or faint pink color develops. If not completely decolorized, add 0.5 g charcoal powder, shake, filter through a coarse filter, and refrigerate in a tightly-stoppered bottle in the dark. Commercially prepared Schiff's reagent can be obtained from several companies (i.e., Fisher Scientific #SS32-500).
