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.     

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.     

Feulgen-Naphthol Yellow S cytophotometry of liver cells. The effect of formaldehyde induced shrinkage on nuclear Naphthol Yellow S binding.

1. In isolated liver cells, fixed in 4 per cent formaldehyde (NFS) for Feulgen-Naphthol Yellow S (F-NYS) staining of DNA and protein, nuclear shrinkage increases the nuclear concentration of solids to 46 per cent (w/v) before the start of the NYS staining. 2. When a fixative mixture of methanol:acetic acid:formalin (85:5:10 by volume; MAF) is used, the concentration of nuclear solids during NYS staining remain at a physiological level of 19 per cent. 3. By exposing liver cells to NFS for 10 to 120 seconds before fixation in MAF, increasing nuclear shrinkage can be induced with increasing pretreatment in NFS. Nuclear NYS binding decreases in parallel with the decreasing nuclear volume in cells thus treated. As the shrinkage induced reduction in NYS binding may vary with the net charge of nuclear non-histone proteins, MAF fixation must be preferred for quantitative determinations of nuclear non-histone protein in F-NYS stained, isolated cells. 4. Fixation in MAF offers the same advantages as NFS fixation as regards the small loss of proteins during the Feulgen staining procedure and the excellent reproducibility of the F-NYS staining. Storage of MAF fixed cells in the fixative for a few days does not alter their F-NYS staining properties. 5. In MAF fixed, F-NYS stained cells there is no NYS binding to histone basic amino acid residues.     

Adaptation of the naphthol yellow S staining for objects with high protein content

Summary In measuring isolated rat liver cells stained with Naphthol Yellow S (NYS) at optimal conditions of pH (2.8), the absorbances measured at the absorption peak of 430 nm appeared to be far too high locally to enable accurate cytophotometric measurements. In order to bring down these absorbances, different techniques for flattening the cells, off-peak measurement and NYS staining at non-optimal pH levels have been applied respectively. Using albumin incorporated in polyacrylamide model films, the reliability of off-peak measurements and the quantitative aspects of the modified protein staining procedures have been investigated. It was found that the NYS procedure can be used as a quantitative protein staining not only at pH 2.8, but also at pH 2.0, 3.5 and 4.0 respectively. The problem with regard to the cytophotometric measuring of isolated liver cells could only be solved, however, by combining a specially developed flattening procedure (by centrifuging small drops of suspension) with staining at non-optimal pH levels. In contrast to the model film results, off-peak measurements applied in situ appeared to give rather unreliable results. In cases of a combined Feulgen-NYS staining, the Feulgen-DNA values were not significantly influenced by any of the modifications of the original NYS staining procedure.     

Adaptation of the Naphthol Yellow S staining for objects with high protein content.

In measuring isolated rat liver cells stained with Naphthol Yellow S (NYS) at optimal conditions of pH (2.8), the absorbances measured at the absorption peak of 430 nm appeared to be far too high locally to enable accurate cytophotometric measurements. In order to bring down these absorbances, different techniques for flattening the cells, off-peak measurement and NYS staining at non-optimal pH levels have been applied respectively. Using albumin incorporated in polyacrylamide model films, the reliability of off-peak measurements and the quantitative aspects of the modified protein staining procedures have been investigated. It was found that the NYS procedure can be used as a quantitative protein staining not only at pH 2.8, but also at pH 2.0, 3.5 and 4.0 respectively. The problem with regard to the cytophotometric measuring of isolated liver cells could only be solved, however, by combining a specially developed flattening procedure (by centrifuging small drops of suspension) with staining at non-optimal pH levels. In contrast to the model film results, off-peak measurements applied in situ appeared to give rather unreliable results. In cases of a combined Feulgen-NYS staining, the Fuelgen-DNA values were not significantly influenced by any of the modifications of the original NYS staining procedure.     

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.     

Feulgen staining of rat liver fixed in different fixatives.

In the present study rat liver pieces fixed in 1) 10 per cent buffered neutral formalin, 2) 4 per cent glutaraldehyde, 3) Heidenhain's-Susa fixative and 4) Flemming's fluid, and following hydrolysis in 1-0 N HC1 at 60degreesC for varying time periods have been stained with the UV Feulgen procedure. The results of this study reveal that following hydrolysis for different time periods the tissue material fixed in formalin show the same staining pattern as those fixed in glutaraldehyde. The material fixed in Heidenhain's-Susa displays an intense Feulgen staining after two different times of hydrolysis, and that fixed in Flemming's fluid shows particular staining intensity for a prolonged time period thus indicating better preservation of DNA than in the materials fixed in the other three fixtatives.     

The use of Light Green and Orange II as quantitative protein stains, and their combination with the Feulgen method for the simultaneous determination of protein and DNA

The protein dyes Light Green and Orange II were studied separately and in combination with the Feulgen-Pararosanilin(SO2) and -Thionin(SO2) method for the simultaneous determination of DNA and protein. - With polyacrylamide modelfilms the pH dependency, specificity and stoichiometry of Light Green and Orange II have been investigated. The results of both staining methods with different biological objects have been compared. - In addition, the Feulgen-Thionin(SO2) method was studied with model films with respect to its specificity and stoichiometry. In biological objects it has been compared with the Feulgen-Pararosanilin(SO2) method. - When combining the Light Green staining with the Feulgen-Pararosanilin(SO2) procedure and the Orange II staining with Feulgen-Thionin(SO2), both Feulgen-DNA stainings, which were first applied, proved to be unaffected by the following protein staining procedure. When the Feulgen procedure was carried out without the dye, followed by Light Green staining, the latter became reduced when a sulfite water rinse was included but was unaffected when a running tap water rinse was used. In the case of the Orange II staining a serious reduction in dye binding capacity was found in both situations. - When the Feulgen-Pararosanilin(SO2) Light Green procedure was carried out on isolated nuclei with all dyes present, a decrease of protein dye binding was observed, similar to that found with the well-known Feulgen-Pararosanilin(SO2) Naphthol Yellow S combination. It is concluded that in spite of this reduction the latter two combinations can be used for the cytophotometric analysis of DNA and protein in the same object.     

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 use of light green and organge II as quantitative protein stains,and their combination with the feulgen method for the simultaneous determination of protein and DNA

Summary The protein dyes Light Green and Orange II were studied separately and in combination with the Feulgen-Pararosanilin(SO₂) and-Thionin(SO₂) method for the simultaneous determination of DNA and protein. — With polyacrylamide modelfilms the pH dependency, specificity and stoichiometry of Light Green and Orange II have been investigated. The results of both staining methods with different biological objects have been compared. — In addition, the Feulgen-Thionin(SO₂) method was studied with model films with respect to its specificity and stoichiometry. In biological objects it has been compared with the Feulgen-Pararosanilin(SO₂) method. — When combining the Light Green staining with the Feulgen-Pararosanilin(SO₂) procedure and the Orange II staining with Feulgen-Thionin-(SO₂), both Feulgen-DNA stainings, which were first applied, proved to be unaffected by the following protein staining procedure. When the Feulgen procedure was carried out without the dye, followed by Light Green staining, the latter became reduced when a sulfite water rinse was included but was unaffected when a running tap water rinse was used. In the case of the Orange II staining a serious reduction in dye binding capacity was found in both situations. — When the Feulgen-Pararosanilin(SO₂) Light Green procedure was carried out on isolated nuclei with all dyes present, a decrease of protein dye binding was observed, similar to that found with the well-known Feulgen-Pararosanilin(SO₂) Naphthol Yellow S combination. It is concluded that in spite of this reduction the latter two combinations can be used for the cytophotometric analysis of DNA and protein in the same object.This work was supported by the Dutch Cancer Foundation Koningin Wilhelmina Fonds grant NUKC 1981-15     

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 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.     

Combined staining of protein-bound sulphydryl groups and DNA in polyacrylamide model systems.

A model system of polyacrylamide films containing protein and DNA has been used to examine the feasibility of combining the dihydroxydinaphthyldisulphide (DDD)-diazonium salt procedure for localizing protein-bound sulphydryl groups with the Feulgen technique for DNA to make possible the direct measurement of both these parameters simultaneously. Optimun conditions for the sulphydryl group reaction require reduction of the protein-containing films in 10% aqueous ammonium sulphide for 3 hr at 50 degree C followed by treatment with a DDD solution at 50 degree C for 4 hr. The final coloured product was developed in a solution of the diazonium salt, Fast Red TR, for 15 min. The azo compound thus produced was completely resistant to hydrochloric acid hydrolysis in the manner of the Feulgen reaction. Calculation of protein-bound sulphydryl groups and DNA from measurements made on doubly-stained films showed excellent agreement between the measured and the expected values.     

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.     

Quantitative cytochemical determination of desoxyribonucleic acid with the Feulgen nucleal reaction

The possibility of using the Feulgen nucleal reaction for a quantitative cytochemical estimation of desoxyribonucleic acid (DNA) was investigated. The intensity of the reaction in nuclei was determined by absorption measurements with the microscope. The accuracy of such measurements was tested by comparison with measurements on the same material with a Beckman spectrophotometer. The values obtained with the microscope agreed within a few per cent with those obtained with the Beckman spectrophotometer. Furthermore, the errors introduced by uneven distribution of absorbing material, by variations in the numerical aperture of the system, and by variation in the area used on the phototube were investigated empirically. The following variables were studied with regard to their effect on the intensity of the Feulgen reaction: type of fixation, time of hydrolysis after acetic acid-alcohol and formalin fixation, time of staining in leucobasic fuchsin, method of preparation of leucobasic fuchsin. The intensity of the Feulgen reaction in liver and erythrocyte nuclei of various vertebrates, fixed in acetic acid-alcohol, was then compared with the DNA content of these nuclei as determined by chemical analysis on a known number of nuclei. The intensity of the reaction was found to be proportional to the DNA content of the nuclei, if nuclei of similar structure and DNA concentration were compared. In nuclei of different structure and DNA concentration (i.e. liver and erythrocyte nuclei), fixed in acetic acid-alcohol, the intensity of the Feulgen reaction was, however, not proportional to the DNA content. This difficulty was overcome by isolating nuclei in sucrose and by fixing them in formalin. Uniform distribution of DNA and therefore uniform coloring after the Feulgen reaction were thus obtained. In such nuclei with uniform distribution of absorbing material the Feulgen reaction was found to be proportional to the DNA content of nuclei, even if they differed greatly in their DNA concentration. The Feulgen nucleal reaction is not quantitative in an absolute sense. For absolute determinations nuclei of known DNA content must be treated together with the unknown material to serve as standard. From these data it therefore appears possible to determine cytochemically relative amounts of DNA in cellular structures by measuring their absorption after treatment with the Feulgen nucleal reaction.     

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.     

A comparative study of quantitative stains for DNA in image cytometry.

In this study we examined the reproducibility of several stains used to measure nuclear DNA by image cytometry. The specimens were touch preparations of liver and testis from mouse and liver, intestine and brain from rat, fixed in either neutral formalin or Carnoy's solution. The tested stains included four Feulgen methods (pararosaniline, azure-A, thionin and acriflavine), the gallocyanine-chromalum stain and two fluorescent stains (acridine orange and propidium iodide). Absorbance measurements employed a video image analysis system; fluorescence measurements were from a scanning microspectrophotometer. The acriflavine-Feulgen stain was analyzed for both absorbance and fluorescence. All seven stains were quantitative for DNA and gave reproducible results. The absorbance measurements had a lower coefficient of variation (CV) than the fluorescence values. In a nested analysis of variance of the pararosaniline Feulgen stains, cell-to-cell variability accounted for 67% of the total variance; slide-to-slide, 9%; and batch-to-batch, 24%. These values did not change significantly when the staining was performed in an automatic staining machine. For DNA analysis using image cytometry, we conclude that the Feulgen staining technique is the most useful. In particular, acriflavine-Feulgen-stained cells fixed in Carnoy's fluid give the least variation between measurement values and the most accurate ratios between the separate ploidy groups. For fluorescence cytometry we recommend Carnoy's fixation and the acriflavine-Feulgen stain because of its narrow CV as compared to acridine orange and propidium iodide.     

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.     

Senescence vs. changes in nuclear DNA, protein and dry mass contents in leaf mesophyll of two species differing in occurrence of endomitotic polyploidy

DNA and Naphtol yellow S-staining (F-NYS) protein contents were measured cytophotometrically using the Feulgen method in the nuclei of the mesophyll from the basal and apical zone of young and old leaves in two perennial monocotyledonous species: Rhoeo discolor and Clivia miniata, differing in presence or absence of DNA endoreplication. Dry mass content was determined interferometrically using an uniform field with large image shearing method. It has been shown that nuclei with 2C DNA and below 2C DNA content dominate in old leaves. The decrease in dry mass content of nuclei correlated with the decrease in NYS protein content. Parallelly a significant increase in NYS protein and DNA contents observed in chromocenters Rhoeo discolor was proportional to the increase in their dry mass. The decrease in nuclear DNA content in mesophyll of old leaves in endoreplicating species was the same as in non-edoreplicating one, however the senescence was more intensive in endoreplicating species.