The Feulgen Reaction for DNA

The Feulgen reaction for DNA was initially described by Feulgen and Rossenbeck in 1924 (42). They found that a mild acid hydrolysis of fixed tissue sections followed by treatment with Schiff's reagent (43) resulted in a dis-

Table 2

Hydrolysis Times in 5 N HCl for Invertebrate Tissues Prepared in Different Fixatives

Table 2

Hydrolysis Times in 5 N HCl for Invertebrate Tissues Prepared in Different Fixatives

Fixative

Hydrolysis

time (min)

Alcoholic fixatives

15-

20

Formaldehyde vapors

20-

25

Formalin fixatives

20-

40

Metallic fixatives (e.g., Cr or Hg)

40-

-60

crete magenta coloring at the sites of DNA in their preparations. Mild acid hydrolysis splits off the purines of DNA so that the residual polynucleotide is identical to apurinic or thymic acid (7,44-46). The removal of purine bases uncovers potential aldehyde groups present in the 2-deoxyribose molecules. Exposure of the aldehyde groups to Schiff's reagent results in the formation of a very stable, highly chromogenic insoluble compound. Variations in the degree of chromatin compaction within a nucleus are also known to affect the number of sites available for DNA staining after acid hydrolysis (7,47-51). Kasten has an excellent review of the use of basic fuchsin analogs in the manufacture of Schiff's reagent to identify DNA and the stoichiometry of the Feul-gen reaction for DNA (52). Despite several extensive studies of dye purity and resultant stain intensity (52-55), the precise mechanism of the Feulgen reaction for DNA is not clearly understood.

Acid hydrolysis removes histone proteins from alcohol-acetic acid fixed chro-matin, and some polymer sugar linkages may also be split during acid hydrolysis. Formalin fixation, however, retains the histone proteins of chromatin, as may be demonstrated by the alkaline fast green technique for basic proteins (56).

1.5.1. Time, Temperature, and Concentration of Acid Hydrolysis are Critical for Successful DNA Staining

Since the introduction of the Feulgen procedure for DNA, HCl has been the hydrolysis agent of choice. Prolonged hydrolysis results in a decrease in staining, presumably caused by chemical alteration, depolymerization, and extraction of the DNA. Acids other than hydrochloric could be used for DNA hydrolysis (phosphoric acid, sulfuric acid, perchloric acid, or trichloroacetic acid). Such a modification to the usual procedure, however, would require empirical cytophotometric determinations for each acid of appropriate concentrations, times, and temperatures for optimal hydrolysis of DNA (55).

The duration of hydrolysis in 5 N HCl at 20-23°C, or the use of the traditional hydrolysis in 1 N HCl at 60°C (see ref. 52), depends, in large part, on the fixation history of the tissue. The values given in Table 2 are estimates for

Feulgen Reaction
Fig. 4. Diagram of molecular structure of the triarylmethane dyes. Pararosaniline is unmethylated. R1( R2, and R3 represent hydrogen atoms that are sequentially replaced by methyl groups during the synthesis of basic fuchsin analogs. (Adapted from ref. 55.)

hydrolysis times in 5 N HCl at room temperature (i.e., 20-23°C) using procedures introduced by DeCosse and Aeillo (57). For each new combination of tissue and fixation procedures, it is necessary to determine appropriate times and temperatures using test tissue samples before processing high-value slide preparations (12,16,55). The presence of aldehyde groups after mild acid hydrolysis can be demonstrated by using aldehyde-blocking agents (58,59).

1.5.2. Schiff's Reagents

Spectrophotometric analyses show remarkably similar absorption curves for pararosaniline and its variously methylated derivatives and their synonyms: Pararosaniline (Magenta 0), Rosaniline (Magenta I), Basic Fuchsin (Magenta II) and New Fuchsin (Magenta III) with no, one, two, and three methyl groups, respectively, as illustrated in Fig. 4.

Pure fuchsin analogs appear to be equally suitable for Feulgen staining, with respect to their staining intensities (see Note 1). All fuchsin dyes have nearly identical absorption curves. There is a shift of about 8 nm when comparing the position of the absorption maxima for Pararosaniline and New Fuchsin (52,53,55). To standardize the Feulgen-Schiff technique, these authors recommend that pure pararosaniline (C.I. 42500) be used to prepare Schiff's reagents (see Note 1). Others workers have reported satisfactory color generation with the use of Magenta II to prepare Schiff's reagent (55). Other dyes, such as Azure A or Azure C, have been used to make so-called pseudo-Schiff reagents, but the chemistry and stoichiometry of these dark blue DNA dye complexes have not been adequately explored (52).

In summary, the Feulgen reaction is not just a "stain" for chromatin like acetocarmine, but it is a specific cytochemical reaction for DNA that is subject

Table 3

Representative Vendors for Basic Fuchsin (C. I. 42500) and/or Pararosaniline (C. I. 42510)

Table 3

Representative Vendors for Basic Fuchsin (C. I. 42500) and/or Pararosaniline (C. I. 42510)

Vendor

Telephone

Website

C and P Sales

(716)634-3061

www.candpsales.com

Fisher Scientific

(800)766-7000

www.fishersci.com

Gallard-Schlesinger

(800)645-3044

www.gallard-schlesinger.com

I. C. N.

(800)654-0530

www.icnbiomed.com

J. T. Baker

(800)582-2537

www.jtbaker.com.

Newcomer Supply

(800)383-7799

www.newcomersupply.com.

Sigma-Aldrich

(800)325-3010

www.sigmaaldrich.com

Thomas Scientific

(800)348-2100

www.thomassci.com

VWR

(800)932-5000

www.vwr.com

to a number of errors, such as differences in (1) the physical state of chromatin domains (i.e., degree of chromatin compaction), (2) preparatory variables such as chemical fixation and "aging" of slides to be compared, and (3) preparatory variables in compounding the Schiff's reagent (12).

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  • galdino
    How to make feulgen reagent?
    8 years ago
  • Sointu
    How to get the DNA number by Feulgers reaction?
    3 years ago
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    What is chemistry of reaction of feulgen reaction?
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