Influence of some aldehyde blocking agents on staining of depurinized DNA with cationic dyes.

Rat liver, spleen and Walker carcinosarcoma imprints were subjected to depurinizing Feulgen hydrolysis and then treated with blocking agents of aldehyde groups. Such blockators as sodium bisulfite and hydroxylamine which multiplay additionally anionic groups in DNA and intensify the reactions with cationic dyes, ensuring anisotropic staining. Hydrazine lowers the binding of carionic dyes to DNA, instead phenylhydrazine, completely blocks both aldehyde and phosphate groups. When the imprints were treated with 2.4-dinitrophenylhydrazine, aldehyde and phosphate groups of apurinic acid were blocked, and DNA staining by cationic dyes occurred only on account of nitrogroups of the blocking agents which have been used. The staining reaction of cationic dyes after the use of anionogenic blocking agents of aldehyde groups is prospective not only for revealing DNA but also for several other compounds with natural or potential aldo- and ketogroups. However the reaction with phenylhydrazine can serve as a staining without removal of DNA prior to staining as an optional procedure.     

Nonspecific ("pseudo-plasmal") dye-binding in the Feulgen nuclear stain and its blocking by azocarmin G

In Feulgen nuclear staining nonspecific dye-binding due to the "pseudo-plasmal reaction" is intensified in isolated cells with intact cytoplasm, and cannot be eliminated by the post-irradiation method. Fluorescence intensity in the cytoplasm sometimes exceeds that of specific nuclear fluorescence, especially in brain and heart muscle cells, and it was almost impossible to perform cytofluorometric DNA quantification on such specimens. Various kinds of aldehyde-blocking agents such as sodium borohydride, 2,4-dinitrophenylhydrazine, aniline, and sodium pyrosulfite were effective in reducing the "pseudo-plasmal reaction". But the blocking effects were not complete because of additional release of reactive aldehyde groups during subsequent Feulgen hydrolysis. Acidic azocarmin G produced a complete block of all "pseudo-plasmal reaction" in acriflavine-Feulgen nuclear staining, allowing accurate DNA-cytofluorometry to be carried out.     

Cytophotometric determination of the binding of basic dyes by DNA following acetylation

Summary DNA was removed from various tissues by histochemical acetylation of amino groups in proteins using pure acetic anhydride, as demonstrated by cytophotometric (UV, Feulgen, gallocyanin chromalum) and biochemical techniques. Since new phosphate groups were simultaneously exposed, the intensity of methylene blue staining was increased in spite of the nucleic acid release. Under conditions where no extraction occurs the staining intensity increases for more than 30 per cent. On the other hand, the staining intensity of gallocyanin chromalum kept constant. As it had been demonstrated previously, that gallocyanin chromalum binds to about 86 per cent of the DNA phosphate groups, it was concluded that this dye binds to a higher percentage of phosphate groups than do the usual basic dyes. Since it is not possible under the conditions used to make all nucleic acid phosphate groups available for basic dye binding by blocking the amino groups of proteins it can be assumed that not only electrostatic, but also spatial and steric relationships influence the binding capacity of basic dyes to the phosphate groups of nucleoproteins.Supported by a grant from the Deutsche Forschungsgemeinschaft, Bad Godesberg, Germany.     

Fluorescence fading and stabilization in cytofluorometry

The factors affecting fluorescence fading in cytofluorometry were investigated using different kinds of nuclear staining, mounting media, and procedure of specimen preparation. Acceleration of fluorescence fading was observed in smear specimens treated with RNase, trypsin, or hypotonic solution before pararosaniline Feulgen nuclear staining. Similar effect was found for other DNA-stainings such as "33258 Hoechst" and Feulgen reactions with different Schiff-type dyes, such as acriflavine-SO2 and cresyl-violet-SO2, when chemically pure DNA was used. Fluorescence decay was rapid for all fluorochromes examined, when glycerin or buffer solution was used as mounting medium. Marked stabilization of fluorescence emission was induced in specimen mounted in non-fluorescent resin, Entellan (Merck), after post-staining fixation with absolute methanol for all tested fluorochromes. The same treatment induced almost complete fluorescence stabilization of fluorescein isothiocyanate (FITC); no detectable fluorescence fading was observed in a specimen stained with indirect immunofluorescence reaction using anti-UV-DNA antibody, during storage for 2 years at room temperature without special protection against light. These observations suggest that factors which bring about conformational stability of macromolecule-dye complexes generally induce fluorescence stabilization.     

The effect of graded 60 degrees C 1N nitric acid extraction and of deoxyribonuclease digestion on nuclear staining by metachrome mordant dye metal salt mixtures.

We can divide metachrome mordant staining of nuclei after graded 60 degrees C 1 N nitric acid extraction into three groups. The Feulgen nucleal reaction and dilute cationic dye staining of nuclei are abolished in about 30 minutes. With one group of metachrome dyes nuclear staining is lost with acid exposures of one hour or less. In a second group nuclear staining is weakened by 30-60 minute extractions, but persists in recognizable grade for 4-6 hours. In the third group nuclear staining remains almost unimpaired for 4-6 hours. In the first group the nuclear staining seems clearly assignable to the nucleic acids and to DNA in particular. In the second group loss of part of the reactivity on short exposure indicates some participation of DNA in the control staining result, as well as participation of basic nucleoprotein. In the third group staining seems assignable largely to basic nucleoprotein. The five gallocyanin group dyes, all in group 1, all possess a dialkylamino group, probably functioning as an ammonium chloride.Hematoxylin, the flurone blacks and gallein all present an o-hydroxysemiquinone group which probably acts as a weak acid, in addition to the carboxyl group of gallein which gives the strongest staining of nuclei at the longest acid exposure. Deoxyribonuclease digestion (2 hours, 37 degrees C) separated sharply a class in which nuclear staining failed completely, a class in which nuclear staining was fully equal to that in the control preparations and an intermediate group in which slight, moderate, or severa impairment was present. Generally there was good agreement between the two methods of nucleic acid removal, despite the fixation difference. In each case, however, the extraction procedure was one worked out for the fixation on which it was used.     

The role of phosphotungstic and phosphomolybdic acids in connective tissue staining I. Histochemical studies

Synopsis An investigation of the role of phosphotungstic and phosphomolybdic acids in Mallory-like trichrome methods showed unexpectedly that, rather than acting as mordants to anionic dyes, these polyacids selectively blocked staining of all tissue components other than connective tissue fibres to Aniline Blue and other similar fibrereactive dyes. Connective tissue components were found to contain residues resembling histidine that are easily accessible to anionic dyes. Blocking towards typical anionic dyes for demonstrating plasma proteins, such as Biebrich Scarlet, was also demonstrated but was less complete. The blockade of both types of dye was labile if the staining times were extended; plasma dyes were more sensitive than fibre dyes in this respect. Histochemical reactions for tyrosine residues were blocked. In connective tissue, phosphotungstic acid did not block histidine residues demonstrable by the coupled tetrazonium reaction with previous iodination. Thus it is postulated that differential trichrome staining occurs by binding of Aniline Blue to basic residues in the connective tissue not blocked by phosphotungstic acid and subsequent replacement of the blocking agent by an anionic dye. The binding of phosphotungstic acid to both epithelium and connective tissue was demonstrated by the quenching of autofluorescence in these regions and by the reduction of the bound PTA to blue coloured products with titanium trichloride.     

Cytofluorometric and cytochemical comparisons of normal and abnormal human cells from the female genital tract.

Acridine orange staining of exfoliated cells from epithelial tissues facilitates discrimination between normal and abnormal cells: abnormal cells develop highly elevated nuclear fluorescence. Comparisons of acridine orange (AO) staining with propidium iodide (PI) or Feulgen staining have shown that: (a) PI staining also provides highly elevated nuclear fluorescence from abnormal cells; (b) the distributions of nuclear fluorescence following AO or PI staining were usually not significantly different as judged by the Kolmogorov-Smirnov test; (c) fluorescence emission spectra from AO and PI stained cells are consistent with the hypothesis that both fluorochromes bind to DNA within cell nuclei; (d) DNAse treatment of AO stained normal cells eliminates the nuclear fluorescence peak from slit-scan contours; RNAse treatment has no effect on nuclear fluorescence; (e) the distribution of abnormal cell nuclear fluorescence after AO staining is usually, but not always, significantly different from the distribution of abnormal cell nuclear absorbance after Feulgen staining, with relative nuclear fluorescence being greater than relative nuclear absorbance. The hypothesis currently most consistent with these results is that elevated Feulgen DNA content can account for only part of the discrimination provided by AO staining, and that the chromatin within abnormal cells is altered so as to increase accessibility of DNA to intercalating dyes.     

The mechanism of action of “mordant” dyes — a study using preformed metal complexes

Summary For small basic dyes blocking or selective extraction of tissue sections can reduce staining (methylation, or trichloracetio acid extraction) or increase staining (acetylation or nitrous acid deamination). This is as predicted by a simple electrostatic model of dye binding. In contrast, the staining with both large basic dyes and with metal complex dyes was only slightly affected by these treatments. This is in keeping with the probable role of van der Waals attractions and hydrophobic bonding in the binding of large dyes.Since MCDs are all large, there is no need to invoke the traditional, special mechanism of mordanting (i.e. tissue-metal ion-dye covalencies) to explain the peculiarities of these MCDs. An apparent exception to this was the resistance of MCDs, unlike either small or large basic dyes, to the action of processing fluids. However, this was due to the insolubility of MCDs in alcoho, lsin contrast to the solubility of most basic dyes, irrespective of size, in these media.     

BUFFERED OSMIUM TETROXIDE (OsO4) FIXATION FOR CYTOLOGICAL AND FEULGEN MICROSPECTROPHOTOMETRIC STUDIES OF HUMAN RECTAL POLYPS

Evidence has been presented that when human rectal mucosa and polyps are fixed with buffered OsO₄ according to the method of Palade, staining of paraffin sections by basic and acidic dyes results in a distinct color difference between chromatin and cytoplasm. Furthermore, the Feulgen reaction also can be carried out successfully and such OsO₄-fixed Feulgen-stained sections are especially suitable for microspectrophotometric determinations of DNA in individual nuclei.     

The Feulgen banded karyotype of the mouse: analysis of the mechanisms of banding

The chromosomes of the mouse have been identified by specific banding patterns revealed by the Feulgen stain. Comparison of the patterns of the Feulgen-stained karyotype with those of acetic-saline-Giemsa stain and quinacrinemustard-fluorescence demonstrates a high order of similarity among the three, with the localization of Feulgen dense bands and regions closely paralleling that of Giemsa dark and fluorescence bright bands. Since the stained substrate of the Feulgen reaction is known to be DNA, it is suggested that all three banding methods reveal the distribution of DNA or of some moiety that closely follows DNA distribution in metaphase chromosomes. The preparative procedure of the Feulgen banding method consists of a 15 to 20 minute exposure to PO₄ buffer at pH 10 and a prolonged (60–72 hrs) exposure to 12xSSC. Omission or curtailment of either step results in preparations with chromosome sets that are not karyotypable, although some stain differentiation is produced. HCl extraction prior to the preparative treatment blocks banding, but acid extraction following the preparative treatment, either that of the HCl hydrolysis of the Feulgen reaction of that of an almost fourfold extension of the standard hydrolysis time, does not obliterate bands already formed. By extrapolation from biochemical studies of chromatin, it is postulated that the localization of Feulgen dark and light stain, representing relative DNA densities, reflects the regional protein association of the DNA; the Feulgen dense regions may result from aggregation of a specific class of histones by the alkaline buffer with consequent condensation of the DNA bound to those histones; the Feulgen pale or negative regions may represent those in which non-aggregated proteins, histone and non-histone, have been solubilized in the saline incubation, rendering the DNA of those regions subject to diffusion or vulnerable to fragmentation in the Feulgen hydrolysis.     

Combined staining procedures for cytophotometry of protein and DNA Feulgen-Naphthol Yellow S and dinitrofluorobenzene-Feulgen

A comparison has been made between dinitrofluorobenzene (DNFB) and Naphthol Yellow S (NYS) as protein stains in combination with the pararosaniline-SO2 Feulgen procedure. Chicken erythrocytes were used as test cells. Cytophotometric measurements were made using a Zeiss scanning stage cytophotometer coupled to a PDP 11/10 minicomputer using the BICOSCAN program to obtain values for protein per cell, protein per "nuclear area' and DNA per nucleus. With 5N HCl as the Feulgen hydrolysis agent, DNFB staining, applied before the Feulgen procedure, was found to be unaffected by hydrolysis conditions required to give optimum Feulgen staining and showed only small losses after longer hydrolysis times. On the other hand measurements of NYS staining, of necessity applied after the Feulgen procedure, seem to be susceptible to the duration of Feulgen hydrolysis. This susceptibility is probably due to the interaction of the DNA phosphates with the basic amino acid residues, potential binding sites for NYS. Since the degree of this interaction may be variable, it is argued that NYS binding will measure the available basicity of proteins at the time of staining but no specific protein fraction. DNFB binding is unaffected by DNA-protein interactions and therefore can give a more reliable measure of "nuclear' protein, particularly in conjunction with Feulgen-DNA measurements.     

The Specificity of the Feulgen Reaction for Thymonucleic Acid

The results of experiments on the specificity of the Feulgen reaction for thymonucleic acid do not substantiate the observations of Carr. The staining is not localized in the nucleus because of the destruction of cytoplasmic constituents following acid hydrolysis or because of the absorbing power of chromatin, since the cytoplasm and nucleolus can still be stained by numerous dyes. The effects of factors such as the acid hydrolysis and sulfurous acid washing baths upon the cytologic distribution of dye were studied on tissues stained with (1) fuchsin-sulfurous-acid (Feulgen) reagent, (2) fuchsin-sulfurous-acid reagent colorized by the addition of formaldehyde, (3) basic fuchsin in one-tenth normal HCl, and (4) basic fuchsin in distilled water. Under comparable conditions, important differences between these stains were found in the effects of preliminary hydrolysis; rapidity of staining and destaining; extractability of dye from tissues by water, alcohol, and sulfurous acid solution; rate of fading from exposure to light; localization of stain in tissues; and differences in hue. After treating tissues with desoxyribonuclease, an enzyme which acts only upon thymonucleic acid, cells do not stain with the Feulgen technic. Following removal of nucleic acid from chromatin by hydrolysis, attempts to demonstrate an absorption of thymonucleic acid upon the residual nuclear protein were unsuccessful.

The evidence for and against the specificity is discussed. In agreement with most other investigators, on the basis of the evidence in the literature as well as these experiments, it is concluded that when properly controlled the Feulgen reaction is relatively specific for thymonucleic acid.     

The Nature of Mycobacterial Acid-Fastness

Phenol is not essential to acid-fast staining, for it will occur in the absence of phenol where such lipoid-soluble basic dyes as night blue, Victoria blue B or Victoria R are used; it is essential for acid-fast staining with water soluble basic dyes such as basic fuchsin. When phenol is added to the staining solution, such water soluble basic dyes behave in effect like their lipid-soluble counterparts. The loss of mycobacterial acid-fastness with carbolfuchsin after bromination or chromation indicates that this phenomenon is related to the presence of unsaturated lipids in the bacterial cells. Within the cells these acid-fast lipids are bound in such a way that they are easily removed from all mycobacteria by hot dilute HCl; from leprosy bacilli alone they are easily removed with hot pyridine. From the results of various blocking reactions it appears that carboxyl and especially hydroxyl groups of these cellular lipids are essential to the acid-fast reaction of mycobacteria.     

The nature of mycobacterial acid-fastness.

Phenol is not essential to acid-fast staining, for it will occur in the absence of phenol where such lipoid-soluble basic dyes as night blue, Victoria blue B or Victoria R are used; it is essential for acid-fast staining with water soluble basic dyes such as basic fuchsin. When phenol is added to the staining solution, such water soluble basic dyes behave in effect like their lipid-soluble counterparts. The loss of mycobacterial acid-fastness with carbol-fuchsin after bromination or chromation indicates that this phenomenon is related to the presence of unsaturated lipids in the bacterial cells. Within the cells these acid-fast lipids are bound in such a way that they are easily removed from all mycobacteria by hot dilute HCl; from leprosy bacilli alone they are easily removed with hot pyridine. From the results of various blocking reactions it appears that carboxyl and especially hydroxyl groups of these cellular lipids are essential to the acid-fast reaction of mycobacteria.     

Fluorescence fading and stabilization in cytofluorometry

Summary The factors affecting fluorescence fading in cytofluorometry were investigated using different kinds of nuclear staining, mounting media, and procedure of specimen preparation. Acceleration of fluorescence fading was observed in smear specimens treated with RNase, trypsin, or hypotonic solution before pararosaniline Feulgen nuclear staining. Similar effect was found for other DNA-stainings such as 33258 Hoechst and Feulgen reactions with different Schiff-type dyes, such as acriflavine-SO₂ and cresylviolet-SO₂, when chemically pure DNA was used. Fluorescence decay was rapid for all fluorochromes examined, when glycerin or buffer solution was used as mounting medium. Marked stabilization of fluorescence emission was induced in specimen mounted in non-fluorescent resin, Entellan (Merck), after post-staining fixation with absolute methanol for all tested fluorochromes. The same treatment induced almost complete fluorescence stabilization of fluorescein isothiocyanate (FITC); no detectable fluorescence fading was observed in a specimen stained with indirect immunofluorescence reaction using anti-UV-DNA antibody, during storage for 2 years at room temperature without special protection against light. These observations suggest that factors which bring about conformational stability of macromoleculedye complexes generally induce fluorescence stabilization.     

The staining behavior of oocyte cytoplasm with the alcoholic Feulgen variant and with methyl green

Summary A variant of the Feulgen reaction which has been proposed as a method for demonstrating cytoplasmic DNA in oocytes has been tested on ovarian material from a variety of species. While Schiff positive staining was developed, this was not removable by pretreatment with DNase and could be reproduced by using oxidants used in the pseudoplasmal reaction. This method was not considered useful for demonstrating cytoplasmic DNA.When chloroform extracted solutions of methyl green were used to stain ovaries, cytoplasmic staining identical in pattern to that obtained with other basic dyes was observed. The cytoplasmic staining was prevented by pretreatment of sections with RNase, but was not affected by DNase pretreatment. In somatic cells with high concentrations of cytoplasmic RNA, only nuclear staining was observed. This nuclear staining was labile to DNase but not to RNase.This work was supported by U.S. Public Health Service grants GM-10003-03 and K-3-6176-03.Contribution number 376 of the Bermuda Biological Station.     

Studies on isolated cell components III. A cytological study of the effects of heparin on isolated nuclei

The gel released from isolated rat liver nuclei in response to heparin treatment has been found to stain with methylene blue, azure A, and methyl green when the dyes were added to the salt-sucrose nuclear isolation medium.Azure A and methylene blue caused rapid nuclear shrinkage to as little as $\text{1}\text{4}$ the original nuclear volume. Subsequent treatment with heparin caused the nuclei to fade rapidly and swell to approximately $\text{5}\text{4}$ of the original volume. With methylene blue stained nuclei heparin caused the extrusion of deeply stained, slightly birefringent rods through apertures on the nuclear surface. Methyl green also caused nuclear shrinkage, but to a lesser degree.Studies with the Feulgen reaction demonstrated structural damage in isolated rat liver nuclei as a result of heparin action. The viscous material released by heparin was shown to be Feulgen positive by resort to hydrolysis without prior fixation, since after customary fixatives the presence of a Feulgen positive reaction outside the nucleus could not be clearly demonstrated. The possibility is suggested that the Feulgen reaction following the customary fixatives depends in part on the manner in which the DNA is bound.The nuclei of leucocytes with visually intact cell membranes included in the nuclear preparations failed to show structural damage due to heparin and it is suggested that either the cell membrane provides some protection against heparin action or that damaged cells are more susceptible to this action.Observations made provide additional basis for the conclusion that heparin replaces DNA in the nucleo-histone of the nucleus, resulting in the structural damage observed, and releasing DNA in the form of a soluble viscous protein containing complex.     

Combined staining procedures for cytophotometry of protein and DNA Feulgen-Naphthol Yellow S and dinitrofluorobenzene-Feulgen

Summary A comparison has been made between dinitrofluorobenzene (DNFB) and Naphthol Yellow S (NYS) as protein stains in combination with the pararosaniline-SO₂ Feulgen procedure. Chicken erythrocytes were used as test cells. Cytophotometric measurements were made using a Zeiss scanning stage cytophotometer coupled to a PDP 11/10 minicomputer using the BICOSCAN program to obtain values for protein per cell, protein per nuclear area and DNA per nucleus. With 5N HCl as the Feulgen hydrolysis agent, DNFB staining, applied before the Feulgen procedure, was found to be unaffected by hydrolysis conditions required to give optimum Feulgen staining and showed only small losses after longer hydrolysis times. On the other hand measurements of NYS staining, of necessity applied after the Feulgen procedure, seem to be susceptible to the duration of Feulgen hydrolysis. This susceptibility is probably due to the interaction of the DNA phosphates with the basic amino acid residues, potential binding sites for NYS. Since the degree of this interaction may be variable, it is argued that NYS binding will measure the available basicity of proteins at the time of staining but no specific protein fraction. DNFB binding is unaffected by DNA-protein interactions and therefore can give a more reliable measure of nuclear protein, particularly in conjunction with Feulgen-DNA measurements.     

The effect of graded 60°C 1N nitric acid extraction and of deoxyribonuclease digestion on nuclear staining by metachrome mordant dye metal salt mixtures

Summary We can divide metachrome mordant staining of nuclei after graded 60°C 1N nitric acid extraction into three groups. The feulgen nucleal reaction and dilute cationic dye staining of nuclei are abolished in about 30 minutes. With one group of metachrome dyes nuclear staining is lost with acid exposures of one hour or less. In a second group nuclear staining is weakened by 30–60 minute extractions, but persists in recognizable grade for 4–6 hours. In the third group nuclear staining remains almost unimpaired for 4–6 hours. In the first group the nuclear staining seems clearly assignable to the nucleic acids and to DNA in particular. In the second group loss of part of the reactivity on short exposure indicates some participation of DNA in the control staining result, as well as participation of basic nucleoprotein. In the third group staining seems assignable largely to basic nucleoprotein. The five gallocyanin group dyes, all in group1, all possess a dialkylamino group, probably functioning as an ammonium chloride. Hematoxylin, the fluorone blacks and gallein all present ano-hydroxysemiquinone group which probably acts as a weak acid, in addition to the carboxyl group of gallein which gives the strongest staining of nuclei at the longest acid exposure. Dexyribonuclease digestion (2 hours, 37°C) separated sharply a class in which nuclear staining failed completely, a class in which nuclear staining was fully equal to that in the control preparations and an intermediate group in which slight, moderate, or severa impairment was present. Generally there was good agreement between the two methods of nucleic acid removal, despite the fixation difference. In each case, however, the extraction procedure was one worked out for the fixation on which it was used. Assisted by National Cancer Institute Contract No. NO 1 CB43912.     

The Feulgen reaction 75 years on

 The Feulgen reaction proposed by Feulgen and Rossenbeck 75 years ago is one of the cytohistochemical reactions most widely used in biology and medicine. It allows DNA in situ to be specifically stained based on the reaction of Schiff or Schiff-like reagents with aldehyde groups engendered in the deoxyribose molecules by HCl hydrolysis. The staining intensity is proportional to the DNA concentration. Current applications of the Feulgen reaction are mainly concerned with DNA quantification in cell nuclei by image cytometry for ploidy evaluation in tumor pathology. From the morphological point of view, specific demonstration of DNA in cell structures at the light microscopic level is very little used nowadays. On the other hand, application of the Feulgen principles to electron microscopy have recently allowed specific DNA-staining procedures to be developed for the study of the structural organization of DNA in situ. Accepted: 13 January 1999