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.     

Exposure and removal of stainable groups during Feulgen acid hydrolysis of fixed chromatin at different temperatures

Summary Hyperdiploid Ehrlioh's ascites tumour cells grown in male mice (strain NMRI) were labeled with radioactive nucleotides. The nucleic acids were extracted from fixed, air-dried smears by fractionated hydrolysis and their radioactivity measured by liquid scintillation. The experiments showed that the exposure of aldehydes through removal of purine bases and the elimination of these aldehydes through depolymerisation of DNA were the two main processes responsible for the Feulgen hydrolysis curve. They were shown to be independent and overlapping. The depurination can be described as a simple hydrolytic reaction, while the extraction of DNA depends on a number of different factors. This entails that, in the Feulgen acid hydrolysis procedure, the part of DNA measured is dependent upon the stability of the chromatin. It was found that it is possible accurately to determine the depolymerisation process and thereby roughly correct the measured amount of Feulgen DNA.     

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.     

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.     

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.     

A study of DNA depolymerisation during feulgen acid hydrolysis

Summary The binding of Schiff dye molecules after acid hydrolysis (1 M HCl) for varying lengths of time was studied in ascites tumour cells. The amount of dye bound to the tumour cells closely followed the number of aldehyde groups, calculated from the extraction of radioactive nucleotides. This constant dye to aldehyde ratio did not change when the hydrolysis was performed at a lower acid concentration (0.3 M HCl). The conclusion drawn is that Feulgen dye measurements represent, in a constant way, the number of aldehydes on DNA at any given time during hydrolysis. The alteration of the hydrolysis pattern of chromatin fixed in formalin was found to be due to a slower extraction of DNA depolymerisation products, the purine liberation being unaffected. A similar explanation is offered for the extreme pattern obtained from hydrolysis of bull spermatozoa chromatin.     

A study of DNA depolymerisation during Feulgen acid hydrolysis.

The binding of Schiff dye molecules after acid hydrolysis (1 M HCl) for varying lengths of time was studied in ascites tumour cells. The amount of dye bound to the tumour cells closely followed the number of aldehyde groups, calculated from the extraction of radioactive nucleotides. This constant dye to aldehyde ratio did not change when the hydrolysis was performed at a lower acid concentration (0.3 M HCl). The conclusion drawn is that Feulgen dye measurements represent, in a constant way, the number of aldehydes on DNA at any given time during hydrolysis. The alteration of the hydrolysis pattern of chromatin fixed in formalin was found to be due to a slower extraction of DNA depolymerisation products, the purine liberation being unaffected. A similar explanation is offered for the extreme pattern obtained from hydrolysis of bull spermatozoa chromatin.     

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.     

Temperature and acid concentration in the search for optimum Feulgen hydrolysis conditions.

Exposure and removal of aldehyde groups during Feulgen acid hydrolysis were studied at a wide range of temperature and acid concentrations. Temperatures between 9 and 75degreesC were found to influence only the rate of the hydrolysis reaction over the entire range from high (6 M) to low (0.05 M) HCl concentrations. The temperature dependence was high, and around +5degreesC was sufficient to double the reaction rate. The influence of acid concentrations between 0.02 and 6 M was studied, and the extraction rates that determine the peak values of the Feulgen hydrolysis curve were found to depend in the same way on the (H+) concentration. A diagram is given that makes it possible to determine the time to reach the point during hydrolysis where the maximum amount of aldehyde groups are developed for a wide range of temperatures and acid concentrations. Temperatures slightly above room temperature in combination with high acid concentration is recommended for Feulgen hydrolysis.     

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.     

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     

Factors affecting the course of DNA acid hydrolysis in carrying out the Feulgen reaction]

Literature data concerning acid hydrolysis of DNA during the Feulgen procedure are reviewed, with emphasis being made on the dependence of Schiff-apurinic acid binding on the fixation technique, the temperature of hydrolysis and acid concentration, the rate of extraction of depolymerized DNA fragments, the nucleotide composition of DNA, the chromatin state, and on the composition of nucleoprotein. Some practical considerations for optimization of the Feulgen procedure for a precise quantitative determination of DNA amount are given.     

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.     

Histochemical properties of spermatozoa and somatic cells

Summary The Feulgen acid hydrolysis patterns of chromatin of different biochemical composition and compactness were analyzed. It was found that the purine extraction rate during acid hydrolysis was affected by the addition of NaCl or 2-mercaptoethanol to the hydrolysis bath. The maximum DNA depolymerization rate was directly correlated to the depurination rate but the extraction rate of hydrolysed DNA was in addition dependent on the stability of the surrounding protein matrix. The results indicate that the diffusion of DNA fragments is partially obstructed in extremely stabilized chromatins (e.g. bull spermatozoa). It is assumed that the extraction pattern of DNA is mainly dependent on the size of the fragments which leave the chromatin by diffusion. It appears that basic proteins do not influence the depolymerization of DNA but there are indications that during certain experimental conditions the purine liberation is dependent upon the chromatin structure.     

Histochemical properties of spermatozoa and somatic cells. III. Depolymerization and extraction of DNA during Feulgen acid.

The Feulgen acid hydrolysis patterns of chromatin of different biochemical composition and compactness were analyzed. It was found that the purine extraction rate during acid hydrolysis was affected by the addition of NaCl or 2-mercaptoethanol to the hydrolysis bath. The maximum DNA depolymerization rate was directly correlated to the depurination rate but the extraction rate of hydrolysed DNA was in addition dependent on the stability of the surrounding protein matrix. The results indicate that the diffusion of DNA fragments is partially obstructed in extremely stabilized chromatins (e.g. bull spermatozoa). It is assumed that the extraction pattern of DNA is mainly dependent on the size of the fragments which leave the chromatin by diffusion. It appears that basic proteins do not influence the depolymerization of DNA but there are indications that during certain experimental conditions the purine liberation is dependent upon the chromatin structure.     

The Feulgen reaction after glutaraldehyde fixation.

A technique is described for performing the Feulgen reaction for DNA on cells and tissues fixed in glutaraldehyde. Blockade free aldehydes by reducing them with fresh 0.5% NaBH4 in 1% NaH2PO4 for 1 hr at room temperature, then rinse in water. Follow by a Feulgen reaction (hydrolysis at room temperature in 6 N HCl for 20 min, Schiff's reagent for 60 min.). Controls assure the completeness and irreversibility of the borohydride blockade. Cytophotometry shows that the DNA content per nucleus is unaffected by the blockade procedure.     

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.     

Feulgen Densitometry: Importance of a Stringent Hydrolysis Regime

Abstract: Feulgen densitometry is still a widely used method for DNA content measurements, but experimental procedures and results are often controversial. The present note is concerned with a recent report in the literature that optimum Feulgen staining required a remarkably longer hydrolysis time with 5 M HCI in Dactylis glomerata L. than in Hordeum vulgare L. (i.e., 62 min versus 20 min at 25 C). As this result is prone to question the usual practice of maintaining unified hydrolysis times for test material and internal standard, we established hydrolysis curves for D. glomerata, H. vulgare, Pisum sativum L. and Allium cepa L. at 20 C and 25C for 0 to 100 min. No striking differences between the species and, in particular, between Doctylis and Hordeum were found. Optimum staining occurred after 60 min with hydrolysis at 20 C and after 25 min at 25 C. It is strongly recommended to conduct the quantitative Feulgen reaction at a precisely controlled temperature instead of an inexact room temperature. The broader plateau of optimum staining at 20 C makes this regime preferable.     

Automated Feulgen staining with a temperature-controlled staining machine

A Shandon Varistain 24-3 staining machine was modified in order to run automated DNA Feulgen staining. Initial studies showed a strict dependence of the staining intensity (integrated optical density [IOD]) on the temperature of the DNA hydrolysis in 4 N HCl: a difference of 0.5 degrees C around the optimum hydrolysis temperature of 27.5 degrees C resulted in IOD differences of up to 7.8% in epithelial cells and up to 12.0% in lymphocytes. A temperature-controlled stainless steel cuvette, covered with a 4 N HCl-resistant material, was developed and integrated into the machine. Temperature measurements were performed at different positions in the cuvette and on glass slides with copper-constantan electrodes fixed on them; no temperature gradient could be detected within the cuvette. The adjusted temperature of 27.5 degrees C remained constant over 24 hours. The coefficient of variation (CV) of the staining intensity in lymphocytes between different areas on the same slide and between different slides of the same staining cycle was less than 0.6%. The CV between different staining cycles was 5.9%. This system for automated Feulgen staining thus gives reproducible and reliable results and may be introduced into routine diagnostic procedures.     

The Feulgen Reaction after Glutaraldehyde Fixation

A technique is described for performing the Feulgen reaction for DNA on cells and tissues fixed in glutaraldehyde. Blockade free aldehydes by reducing them with fresh 0.5% NaBH₄ in 1% NaH₂PO₄ for 1 hr at room temperature, then rinse in water. Follow by a Feulgen reaction (hydrolysis at room temperature in 6 N HCI for 20 min, Schiff's reagent for 60 min.). Controls assure the completeness and irreversibility of the borohydride blockade. Cytophotometry shows that the DNA content per nucleus is unaffected by the blockade procedure.