A brief history of the Feulgen reaction

Abstract

One hundred years ago, Robert Feulgen published a landmark paper in which he described the first method to stain DNA in cells and tissues. Although a century has passed since the discovery by Feulgen and Rossenbeck, the chemical reaction still exerts an important influence in current histochemical studies. Its contribution in diverse fields, spanning from biomedicine to plant biology, has paved the way for the most significant studies that constitute our current knowledge. The possibility to specifically explore the DNA in cell nuclei while quantifying its content makes it a contemporary and timeless method. Indeed, many histocytochemical studies following the 1924 paper have led to a deep understanding of genome organization in general as well as several specific mechanisms (e.g. DNA duplication or tumour pathology) that, nowadays, constitute some of the most fundamental pillars in biological investigations. In this review, we discuss the chemistry and application of the Feulgen reaction to both light and electron microscopy.

Keywords: Electron microscopy; Feulgen reaction; Light microscopy; Osmium ammine; Schiff-type reagent.

Fig. 1

a Schematic representation of a DNA nucleotide in an acidic environment. Hydrogen ions mediate the detachment of guanine, therefore leaving the DNA nucleotide apurinated (readapted from Pourshahian 2021). b Typical Feulgen hydrolysis curve. The x-axis represents the time required for hydrolysis and the y-axis indicates the Feulgen–DNA values. The plateau corresponds to the maximum level of DNA depurination while the ascending and descending branches represent DNA depurination and depolymerisation, respectively (modified from Mello and Vidal 2017). c One of the possible interpretations of the chemical reaction occurring when the Schiff reagent interacts with aldehyde groups. According to this hypothesis, the aldehydic group forms an alkylsulfonic acid when interacting with SO₂ groups; the binding of the C atom from the alkyl group with the primary aromatic amine re-establishes the chromophoric function of the dye, consequently producing a magenta staining (modified from Hubbe et al. 2019)

Fig. 2

HeLa cells in mitosis after Feulgen reaction and observed under phase contrast. a Anaphase; b Telophase and late telophase. Courtesy of Carlo Pellicciari. Bar = 10 µm

Fig. 3

From Alain Gautier’s archives. The text reads: Metallic polyam(m)mines. Reagents tested for a Schiff-type or Luft-type specificity. Diamino dinitrite palladium; methylamine tungstate; osmium ammine complexes. As shown, palladium and tungsten are weakly positive or negative, while osmium ammine results were positive in all the different batches utilized. The exception is represented by Osmeth (osmium methenammine) and hexaammineosmium triiodide

Fig. 4

a P815 mouse mastocytoma cell, immunolabelled for nucleolin and stained for DNA with OA-B. HCl hydrolysis 45 min, OA-B staining 60 min. Note the intense nucleolar labelling and the highly contrasted DNA. cy: cytoplasm; nc: nucleolus. Bar = 500 nm. b P815 cell, OA-B staining. At high magnification note the fibrillar centre (asterisk) and thin filament of DNA emerging from the condensed DNA region the region. chr: condensed chromatin at the periphery of the nucleolus. Bar = 100 nm