Optimization of the histochemical demonstration of DNA using 3-hydroxy-2-naphthoic acid hydrazide and fast blue B

Previous methods for the histochemical demonstration of DNA were optimized. p-Toluene sulfonic acid as catalyst for hydrazone formation between the aldehydes generated after Feulgen hydrolysis and 3-hydroxy-2-naphthoic acid hydrazide (NAH) was used instead of acetic acid. Modifications of the conditions of the coupling reaction with Fast Blue B reduced the background staining. The optimized histochemical staining method for DNA (NAH-FB-DNA staining) can be performed easily and reproducibly. Without prior Feulgen hydrolysis the optimized method can also be used for the histochemical demonstration of reactive carbonyls undissolved under the given histochemical conditions.     

Quantitative cytochemical aspects of a combined Feulgen-Naphthol Yellow S staining procedure for the simultaneous determination of nuclear and cytoplasmic proteins and DNA in mammalian cells

The simultaneous cytophotometric determination of nuclear and cytoplasmic proteins and DNA by means of a combined Feulgen-Naphthol Yellow S (NYS) staining procedure was investigated. According to this procedure Feulgen staining is performed prior to NYS staining. The following main results were obtained:

1. 1. After NYS staining alone, the amount of NYS bound to the cell was found to be closely correlated to the cellular dry mass. The correlation coefficient was 0.99 in ethanol-acetone fixed cells and 0.95 in formaldehyde-fixed cells. This close correlation was not significantly altered by the Feulgen staining procedure and was 0.92 in ethanol-acetone and 0.94 in formaldehyde-fixed cells. However, the absolute amount of NYS bound per unit dry mass was affected by the method of fixation and type of Feulgen hydrolysis.

2. 2. The cells lose material during the Feulgen procedure, particularly during the acid hydrolysis stage. The type of hydrolysis most suitable for the Feulgen procedure (5 N HCl, 22 °C, 60 min) resulted in a considerable loss of dry mass in ethanol-acetone fixed cells. This loss was smaller in formaldehyde-fixed cells (15%) and was in addition closely correlated (correlation coefficient 0.99) to the dry mass of the cells prior to hydrolysis. In formaldehyde-fixed cells the dry mass after the Feulgen procedure is thus a good measure of the true cellular dry mass of the fixed cells. This is further demonstrated by the close correlation between NYS binding to Feulgenstained cells and the dry mass of these cells prior to the Feulgen procedure (correlation coefficient 0.95).

3. 3. When using the combined Feulgen-NYS staining procedure under standardized conditions (formaldehyde fixation and acid hydrolysis in 5 N HCl, 22 °C, 60 min) a constant amount of NYS was found to be bound per unit dry weight to nuclear and cytoplasmic proteins in various types of mammalian cells with different proliferative activity.

4. 4. The Feulgen DNA determination was not found to be quantitatively affected by the subsequent NYS staining.

From the results of the present study it seems that, under standardized conditions, the combined Feulgen-NYS staining procedure can be used as a reliable quantitative method for the determination of nuclear and cytoplasmic proteins and DNA in mammalian cells.     

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.     

Feulgen Reaction for Thymonucleic Acid

The importance of thymonucleic acid in tissues is discussed briefly. The technic of the Feulgen reaction which has been employed in photometric histochemical observations in tumors is described. The evidence for the specificity of the Feulgen reaction is reviewed and additional experimental observations are reported. The staining of tissues by the Feulgen reaction is compared with that of hematoxylin, basic fuchsia, and fuchsin-sulfurous-acid reagent in which the color had been developed by the addition of formaldehyde. The stains were compared with respect to (1) the selective staining of the cytologic components of the tissues, (2) the staining of tissues following varying intervals of acid hydrolysis and (3) the photometric determination of the fading of the stained tissue by a carbon arc light. The photometric apparatus employed is suitable for the study of many problems on the staining of tissues. Staining by the Feulgen reaction is different from that of both the basic fuchsin from which the fuchsin-sulfurous-acid was prepared and from that of the product of the fuchsin-sulfurous-acid which had reacted with an aldehyde. Under carefully controlled conditions, the Feulgen technic is a relatively specific histochemical reaction for thymonucleic acid.     

The effect of HCl hydrolysis on the retention of thymidine in DNA

Allium roots grown in C(14)-thymidine and H(3)-thymidine media were treated with N hydrochloric acid at 60 degrees C. as in standard Feulgen hydrolysis. The retention of the radioactive thymidine in DNA as a function of hydrolysis time was studied autoradiographically. No significant loss of label was detected until hydrolysis was extended beyond the optimal time for Feulgen staining. The data are consistent with the assumption that there is no significant loss of DNA during normal Feulgen hydrolysis in the material used.     

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 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     

Estimation of residual DNA in Feulgen reaction. A new correction of DNA determination per nucleus.

For the determination of the residual DNA amount after acid hydrolysis of Feulgen's method, a high salt-fluorochrome assay for DNA (5 microM Hoechst 33258 with 1 M NaCl) was effectively applied. At an optimal time length of acid hydrolysis for Feulgen reaction, the ratio of the residual DNA of non-hydrolysis to total DNA is 10% or more in hepatocyte or lymphocyte nuclei. A lot of residual DNA seems not to be negligible in Feulgen's method. A more accurate determination of DNA can be made by correcting the loss ratio of the residual DNA value to Feulgen DNA value. Thus, the combination assay of Feulgen's method with the present fluorometry is enough to measure separately both the amounts of Feulgen DNA and its residual DNA and successfully determines more accurately the total DNA per nucleus by summing both the amounts. The residual DNA, a resistant portion of the chromatin DNA against acid hydrolysis, is a possible constituent as the physiological component of nuclear structures.     

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.     

Quantification of nuclear DNA and intracellular glycogen in a single cell by fluorescent double-staining

Summary It was found that intracellular glycogen is stabilized against acid treatment when it is stored under dry conditions for three months after methanol fixation. This stabilization allowed quantitative double fluorescence staining, for nuclear DNA and intracellular glycogen, in a single cell. A Feulgen nucleal reaction with acriflavine-Schiff's reagent following 5 N HCl hydrolysis at 25°C for 4 min, was followed by a pararosanilin-Schiff PAS reaction for glycogen. This short term hydrolysis was found to be sufficient for the performance of a acriflavine-Schiff's Feulgen nucleal reaction and to provide good preservation of intracellular glycogen. Quantification of nuclear DNA and intracellular glycogen was consecutively carried out with a digital microfluorometer on a single ascites cancer cell of the AH-13 line stained by this method. It was found that there is a positive linear correlation between the amount of DNA and glycogen in this cell line.This work was partly supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, Japan     

Quantification of nuclear DNA and intracellular glycogen in a single cell by fluorescent double-staining.

It was found that intracellular glycogen is stabilized against acid treatment when it is stored under dry conditions for three months after methanol fixation. This stabilization allowed quantitative double fluorescence staining for nuclear DNA and intracellular glycogen, in a single cell. A Feulgen nucleal reaction, with acriflavine-Schiff's reagent following 5 N HCl hydrolysis at 25 degrees C for 4 min, was followed by a pararosanilin-Schiff PAS reaction for glycogen. This short term hydrolysis was found to be sufficient for the performance of a acriflavine-Schiff's Feulgen nucleal reaction and to provide good preservation of intracellular glycogen. Quantification of nuclear DNA and intracellular glycogen were consecutively carried out with a digital microfluorometer on a single ascites cancer cell of the AH-13 line stained by this method. It was found that there is a positive linear correlation between the amount of DNA and glycogen in this cell line.     

Rapid method to obtain high quality nuclear and chromosome images directly from living cells in the Characeae

This study focused on a method based on the capacity of cationic dyes to stain only the nucleus and chromosomes in cells subjected to either acid or alkaline hydrolysis. The method and the squash were optimized for the Characeae and were described in detail. Nuclei from vegetative shoot apices and antheridial filament cells of unfixed, fixed and herbarium material of Nitella opaca were investigated using the Azure A or Toulidine Blue stains. Comparisons with some other staining methods, used in the previous studies, were also reported. The Azure A/Toulidine Blue method is useful to obtain clear images of chromosome morphology comparable or higher to that obtained with Feulgen or Aceto‐Orcein. It requires little time and a less complicated procedure in comparison with other staining methods.     

Immunocytochemical detection of sulphonylated DNA in tissue sections

Summary We report here on a new sensitive and highly specific DNA staining technique which we have called sulpho-DNA staining. DNA staining is based on a sulphonylation reaction of 2-deoxycytidine or cytidine that takes place in the 6th position of cytosine with ensuing immunodetection of the sulphonylated DNA. The specificity of DNA staining is introduced by the use of an antibody recognizing only modified DNA but not modified RNA, by recourse to an additional acid hydrolysis step which destroys RNA but not DNA. We describe here the optimal conditions for the sulphonylation of DNA using O-methylhydroxylamine and metabisulphite as reactants. The new DNA stain labels all nuclei in either normal human tissue or in tumor cells. For nuclear DNA the staining signal is higher for the sulpho-DNA staining than for the Feulgen staining for nuclear DNA. This new DNA staining technique is suitable for use on tissue sections as well as on cytosmears.     

Differentiation of Nucleic Acids by Staining at Controlled pH and by A Schiff-Methylene Blue Sequence

Fixation with Bouin's fluid preserves cytoplasmic and nucleolar ribonucleic acid (UNA) particularly well. RNA may be demonstrated preferentially in Bouin fixed tissue by staining with 0.02% thiazine dye in aqueous McIIvaine phosphate-citrate buffer between pH 3 and 4. Methylation blockage of basophilia other than that of nucleic acids permits staining of RNA with thiazine dyes near neutrality. The deoxyribonucleic acid (DNA) of chromatin undergoes a Feulgen type hydrolysis in the tissue block during 24 hr fixation with Bouin's fluid. This hydrolysis by picric acid permits Schiff staining of the DNA wthout further acid hydrolysis. Consequently after Bouin fixation it is possible to demonstrate DNA and RNA specifically by a Schiff-methylene blue sequence. Thus a Schiff stain without further acid hydrolysis followed by 0.02% methylene blue in phosphate-citrate buffer at pH 3.0 to 3.5 colors DNA magenta in contrast to the blue of RNA.     

The cytochemical localization of lysozyme in Paneth cell granules

Synopsis The breakdown products resulting from the hydrolysis of chitin by lysozyme stain with Alcian Blue. A method based upon this observation has been developed for the histochemical demonstration of lysozyme activity. The application of this method to the jejunal crypts of several animal species indicates that Paneth cell granules contain lysozyme. The binding of the hydrolysis products with Alcian Blue is so strong that any other alcianophilia (e.g. of acid mucosubstances in goblet cells) can be removed selectively by washing withN-cetylpyridinium chloride and counterstaining with Basic Fuchsin and Nuclear Fast Red.     

An efficient histochemical method for deoxyribonucleic acids using a silver enhancement procedure

Summary An efficient histochemical method has been worked out for the demonstration of deoxyribonucleic acids (DNA) with light microscopy. This involves a hot hydrochloric acid-thiocarbohydrazide-silver proteinate sequence followed by a physical development procedure. When applied to tissue sections from rabbit organs such as the spleen, cerebellum and testis, the new method has given excellent discrimination of nuclear DNA in all cells examined. Thus, the method is higher in efficiency and visibility of reaction products than the traditional hot HCl-Schiff (Feulgen) method used hitherto. Its specificity is also high.     

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.     

Improved Histology for the Chick-Quail Chimera

A simple modification of nuclear staining after acid hydrolysis has been made which provides easy identification of quail nuclear markings in a chick-quail chimera. This method also improves the histologic detail normally seen with hematoxylin and eosin when compared to the more commonly used Feulgen reaction. Embryonic tissues can be fixed in Zenker's or Helly's solution and the sections obtained are hydrolyzed in acid (3.5 N HCl at 37 C for 40-50 min). After acid hydrolysis the sections are stained with hematoxylin and eosin rather than Schiff reagent and fast green. The interphase nuclei of chick cells show homogeneous or mottled purplish blue staining, while quail nuclei contain a dark blue spot. This staining corresponds to the reddish purple staining of the quail's heterochromatin seen adjacent to the nucleolus in the standard Feulgen stain. This new technique facilitates identification of quail cell types in the chick host and provides superior histology of the chick tissues by demonstrating cytoplasmic detail.     

Standardization of the Feulgen reaction: the influence of chromatin condensation on the kinetics of acid hydrolysis.

The purpose of the present study was to investigate the influence of chromatin compactness on the kinetics of acid hydrolysis in the Feulgen reaction in cytology. Tissue imprints of rabbit liver, of human bronchial carcinoma and of human blood smears, fixed with alcohol, formaldehyde or with B?hm's solution with and without prior air drying, were stained with a standardized pararosanilin-Feulgen reagent. The time for hydrolysis varied between 7.5 and 120 min. The integrated optical density (IOD) of the cell nuclei was measured with an image analyzer (IBAS 2000). Cells with condensed chromatin (lymphocytes, small cell carcinoma, formaldehyde fixed cells) showed a slow increase of staining intensity and late plateau phase as compared with cells with decondensed chromatin. DNA in condensed nuclei was less susceptible to acid hydrolysis. The degree of chromatin compactness which determines the sensitivity of DNA to hydrolysis is influenced by the type of fixation, cell type and by the functional status of the cell. The conclusion is that Feulgen staining intensities of cells with different degrees of chromatin compactness cannot be compared unless measured in the respective plateau phases of the relevant hydrolysis curves which must be determined individually for each cell type.     

The histochemical demonstration of aniline hydroxylase activity in rat liver

Synopsis A procedure is described for the histochemical demonstration of aniline hydroxylase activity in cryostat sections of rat liver. Tissue sections are incubated in a medium containing aniline; thep-aminophenol formed as a result of enzymatic action is coupledin situ with Fast Blue RR. The staining reaction is found to be confined to the cytoplasm of the hepatocytes. Confirmatory tests for true enzymatic staining reaction include the incubation of sections in medium from which aniline is omitted, and under conditions of enzyme inhibibition. A method for the quantitation of the histochemical staining reaction is also described.The histochemical reactions have been investigated on rat livers subjected to conditions eliciting microsomal enzyme stimulation and inhibition, bothin vitro andin vivo. A close correlation was found between the staining reactions observed and the results of the quantitative histochemical method and the biochemical estimations of aniline hydroxylase activity in liver microsomal fractions obtained by differential centrifugation.