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.     

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.     

MEASUREMENTS OF SOME CELLULAR CHANGES DURING THE FIXATION OF AMPHIBIAN ERYTHROCYTES WITH OSMIUM TETROXIDE SOLUTIONS

On average, 15 per cent of the total haemoglobin present in the blood of the newt Triturus cristatus was extracted during 45 minutes of fixation in Palade-Caulfield fixative. This extraction was reduced with fixatives buffered at pH 6.2 instead of pH 7.4. The addition of Ca⁺⁺ ions to a final concentration of 0.01 M in the fixative completely suppressed haemoglobin extraction. The effect of the pH, and the presence or absence of Ca⁺⁺ ions in the fixative, on the rate of haemoglobin extraction has been determined. During Palade-Caulfield fixation the average projected area of newt erythrocytes increased by 37 per cent, and after dehydration and embedding in Epon the average area was 25 per cent greater than that of the unfixed cell. Fixatives buffered at pH 6.2 and containing 0.01 M Ca⁺⁺ ions caused cellular shrinkage, with the average projected area decreasing by 10 per cent in the fixative. This shrinkage continued during dehydration, and the final average area of the erythrocytes in Epon was 26 per cent less than that of the unfixed cells. Similar measurements with erythrocytes of Amphiuma tridactylum showed that after Palade-Caulfield fixation the average cellular area was increased by 45 per cent, and after dehydration and embedding in Araldite it was 36 per cent greater than that of the unfixed cell. The average nuclear area increased by 35 per cent during fixation but after embedding it was 26 per cent greater than that of the unfixed nuclei. With a fixative at pH 6.2 containing 0.01 M Ca⁺⁺ ions, both the nucleus and the whole cell shrank during fixation. The nuclear area decreased by 20 per cent and the cellular area by 22 per cent. After dehydration and embedding in Araldite, the average nuclear area had decreased by 35 per cent and the cellular area by 40 per cent. It has been shown that OsO₄ fixation lowers the isoelectric points of haemoglobins and other proteins. This finding has been used in the interpretation of the observed cellular changes resulting from fixation.     

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.     

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 LOCALIZATION OF ALBUMIN AND FIBRINOGEN IN HUMAN LIVER CELLS

Human liver sections were stained with anti-human serum albumin and/or anti-human fibrin monomer fluorescent conjugates. Approximately 10 per cent of the hepatic cells stained specifically for human serum albumin,1 per cent for fibrinogen, and 0.1 per cent for both. Approximately 18 per cent of the Kupffer cells stained specifically for human serum albumin and 33 per cent for fibrinogen. Staining of both cell types was mainly cytoplasmic, although albumin was found in the nuclei of some parenchymal cells, depending on the method of fixation. Cytoplasmic granules staining specifically for fibrinogen could be seen just inside the cell membrane facing the bile caniculi in many more parenchymal cells than the 1 per cent showing diffuse cytoplasmic staining. The technical difficulties involved in preparing fluorescent conjugates against these antigens and in the fixation of these antigens for immunofluorescent staining are discussed.     

Porcine Intestinal Mast Cells. Evaluation of Different Fixatives for Histochemical Staining Techniques Considering Tissue Shrinkage

Staining of mast cells (MCs), including porcine ones, is critically dependent upon the fixation and staining technique. In the pig, mucosal and submucosal MCs do not stain or stain only faintly after formalin fixation. Some fixation methods are particularly recommended for MC staining, for example the fixation with Carnoy or lead salts. Zinc salt fixation (ZSF) has been reported to work excellently for the preservation of fixation-sensitive antigens. The aim of this study was to establish a reliable histological method for counting of MCs in the porcine intestinum. For this purpose, different tissue fixation and staining methods that also allow potential subsequent immunohistochemical investigations were evaluated in the porcine mucosa, as well as submucosa of small and large intestine. Tissues were fixed in Carnoy, lead acetate, lead nitrate, Zamboni and ZSF and stained subsequently with either polychromatic methylene blue, alcian blue or toluidine blue. For the first time our study reveals that ZSF, a heavy metal fixative, preserves metachromatic staining of porcine MCs. Zamboni fixation was not suitable for histochemical visualization of MCs in the pig intestine. All other tested fixatives were suitable. Alcian blue and toluidine blue co-stained intestinal goblet cells which made a prima facie identification of MCs difficult. The polychromatic methylene blue proved to be the optimal staining. In order to compare MC counting results of the different fixation methods, tissue shrinkage was taken into account. As even the same fixation caused shrinkagedifferences between tissue from small and large intestine, different factors for each single fixation and intestinal localization had to be calculated. Tissue shrinkage varied between 19% and 57%, the highest tissue shrinkage was found after fixation with ZSF in the large intestine, the lowest one in the small intestine after lead acetate fixation. Our study emphasizes that MC counting results from data using different fixation techniques can only be compared if the respective studyimmanent shrinkage factor has been determined and quantification results are adjusted accordingly.Key words: mast cell, swine, fixation, tissue shrinkage factor     

Quantification of nuclear non-histone proteins by Feulgen-Naphthol Yellow S cytophotometry

Summary Owing to the accumulation of nuclear non-histone protein (NHP) (a) in cells entering the cell cycle from the quiescent state and (b) in continuously cycling cells during G₁ phase, a simultaneous determination of DNA and nuclear NHP is of high potential utility in cell kinetic studies. This paper provides guidelines for a Feulgen-Naphthol Yellow S staining technique for this purpose. It discusses details of the preparation and quantification procedures, and reviews the evidence for a quantitative relationship between nuclear Naphthol Yellow S binding and nuclear NHP.     

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.     

Diurnal variations in endogenous RNA polymerase activity and amounts of nuclear non-histone protein, DNA and cytoplasmic protein in rat liver.

From rats fed ad libitum and kept under a 12 + 12 h light/dark regimen, the DNA dependent RNA polymerase activity of liver cell nuclei was determined avery four hours. From identical rats, nuclear non-histone protein and DNA, and cytoplasmic protein was determined by Feulgen-Naphthol Yellow S cytophotometry of isolated liver cells. The minimum: maximum ratio of the RNA polymerase activity is 0.77; the min:max ratio of nuclear non-histone protein is 0.84. These two parameters have identical time courses with a gradual decline during the light period and a sharp rise after the onset of the dark period. The variations in nuclear DNA content, estimated as the amount of Feulgen stain bound, closely parallel those of the RNA polymerase activity and nuclear non-histone protein content (min:max = 0.96). The amount of cytoplasmic protein per cell also varies throughout the day, but its time curve lags behind those of nuclear non-histone content and RNA polymerase activity. These results are consistent with the concept of nuclear non-histone proteins as de-repressors of the DNA template in differentiated, non-proliferating cells, and support the validity of using Feulgen-Naphthol Yellow S cytophotometry of nuclear non-histone proteins as an estimate of gene expression in such cells.     

The importance of tissue fixation for light microscopic immunohistochemical localization of peroxisomal proteins: the superiority of Carnoy's fixative over Baker's formalin and Bouin's solution

We have compared the effects of fixation with three commonly used fixatives upon preservation of the antigenicity of six peroxisomal proteins in rat liver using both immunohistochemical staining and Western blotting of fixed tissue extracts. The immunoreactivity of all six peroxisomal proteins was well preserved and peroxisomes were clearly identified in material fixed in Carnoy's fixative. Moreover, the corresponding proteins stained well in Western blots prepared from extracts of Carnoyfixed material. The intensity of the immunohistochemical staining was reduced at different rates for individual peroxisomal proteins after fixation in Baker's formalin, but peroxisomes were still well visualized with antibodies to catalase and some -oxidation enzymes. No evidence of immunohistochemical staining for any peroxisomal antigens was obtained after fixation in Bouin's fluid. For detection of the antibody binding sites in Carnoy's fixed material, the avidin-biotin-peroxidase complex (ABC) with aminoethyl carbazole as chromogen was found to be superior to the methods of peroxidase-antiperoxidase/diaminobenzidine and protein A-gold with silver intensification. Using Carnoy-fixative and the ABC-method, we demonstrate light microscopic immunohistochemical localization of peroxisomal antigens in several rat tissues as well as in human post-mortem liver.     

Effects of Fixation on Cell Volume of Marine Planktonic Protozoa

The effects of fixation on the cell volume of marine heterotrophic nanoflagellates and planktonic ciliates were investigated. Decreases in cell volume depended on the combination of the protozoan taxa and the particular fixative. For a particular fixative and protozoan species, degree of shrinkage was independent of physiological state. The volume of fixed cells was found to be approximately 20 to 55% lower than the cell volume of live organisms. For the heterotrophic microflagellates, the fixatives ranked, in order of decreasing effect on cell volume, as glutaraldehyde, formaldehyde, acid Lugol's solution, and modified van der Veer solution. With oligotrichous ciliates and a tintinnid ciliate, formaldehyde caused less shrinkage than glutaraldehyde or acid Lugol's solution. With the aldehyde fixatives, the microflagellates were found to shrink more than the ciliates. Differential effects of fixation on cell volumes may result in an underestimation of the biomass of certain protozoan taxa in natural samples.     

Evaluation of fixative composition,fixative storage,and fixation duration on the fine structure and volume of root-cell nucleoli

Summary— The effect of various combinations of three fixative compositions (glutaraldehyde buffered in veronal acetate, cacodylate, and piperazine-N, N'-bis[2-ethanesulfonic acid]—PIPES], two fixative storage times (fresh vs 6 weeks), and two fixation durations (3 h vs 9 days) on nucleolar fine structure and nucleolar volume in three root cell-types of oat seedlings (Avena sativa L, cv Seger) were evaluated. All fixatives show overall good preservation of fine structure. Nucleolar components are distinct and well delineated in cells fixed in solutions buffered with either cacodylate or veronal acetate; the components are more condensed when preserved in fixative buffered with PIPES. Nucleolar volume is greatest in cells fixed in the cacodylate fixative, and smallest in those preserved in the PIPES fixative. Among the treatments tested, the PIPES fixative evidently best maintains nucleolar volume. Distracting particulate deposits are abundant on nuclei and nucleoli in cells preserved in the veronal-acetate fixative. Contrary to common assumptions, aging of buffered fixative at room temperature for 6 weeks seems to affect neither the general quality of cellular preservation nor the pH of the fixatives, although nucleolar volume is reduced by such treatment. Long-period fixation (9 days) results in destruction of membrane integrity (mitochondria, plastids, ER), and shrinkage of organelles from the cytoplasm. Nucleolar volume is reduced with prolonged fixation.     

Binding of fluorescein isothiocyanate conjugated lectins to rat spermatogenic cells in tissue sections

Summary The testes from three months old Spague-Dawley rats were fixed in Bouin's fluid or neutral buffered 10% formalin, embedded in paraffin, sectioned and after deparaffination stained with the following fluorescein isothiocyanate coupled lectins: PNA, WGA, Con A, RCA, SBA, DBA and UEA. The results show that there are considerable differences in the staining pattern of various spermatogenic cells between different lectins. The fixation in Bouin's fluid enhanced the staining of all the lectins compared to formalin fixation in which only a weak staining could be seen in the acrosomes of spermatids after WGA or PNA staining. PNA and WGA stained specifically the acrosome of the developing spermatids, which was seen from the beginning of the acrosome formation and lasted up to late spermiogenesis. However, the staining with PNA decreased in the late spermatids whereas the intensity of the staining remained unchanged with WGA. Con A did not stain the acrosome but stained unspecifically the cytoplasm of all spermatogenic cells. RCA stained faintly the acrosome throughout the spermatid differentiation. DBA and UEA stained specifically the chromosomes of B spermatogonia. DBA also faintly stained the cell membranes of early spermatids. SBA did not show any specific staining of the spermatogenic cells. Based on this it is suggested that the carbohydrates and glycoproteins which are known to be present in the acrosome are formed already in the beginning of the acrosome formation. The decrease in the PNA staining in late spermatids possibly reflects the fact that the receptor molecules are not synthesized in late spermatids but are formed in earlier developmental stages and are thereafter preserved in the acrosome. The enhancement of lectin binding caused by Bouin's fixative might also be applied to other tissues where previous experiments with formalin fixed tissue have failed to show any staining.     

Human ferritin: effects of antigen source and fixation on leucocyte staining by immunoperoxidase technique

The localization of ferritin was studied in peripheral blood cells and variously fixed tissues with the antibodies against ferritins isolated from human heart and spleen. The unlabelled antibody enzyme method (PAP) was used to detect the binding sites of antibodies. In peripheral blood cell smears both antisera gave rise to strong staining of polymorphonuclear (PMN) cell cytoplasm, whereas the monocytes stained relatively weakly. There were no staining differences between the two antisera. In human spleen sections the spleen ferritin antiserum stained the PMN cells and sinusoidal lining cells, whereas the heart ferritin antiserum stained only PMN cells. Neither of the two antisera stained monocytes in the spleen sections. This finding was observed in specimens fixed in Bouin's fixative, Baker's fixative and neutral formalin. However, the immunoreactivity of ferritin was totally destroyed by some other fixatives (Carnoy's fixative, formol sucrose and glutaraldehyde). These results suggest that ferritin is more readily released from monocytes than from PMN cells, and that mature spleen macrophages contain antigenic determinants of ferritin that are recognized only by anti-spleen ferritin antiserum.     

Tissue fixation with diimidoesters as an alternative to aldehydes. II. Cytochemical and biochemical studies of rat liver fixed with dimethylsuberimidate.

Rat liver fixed with dimethylsuberimidate (DMS) was studied to investigate the use of diimidoesters as dixatives for light and electron microscopic cytochemistry. Paraffin sections of liver fixed with DMS at pH 9.5 were weakly stained with the ninhydrin-Schiff procedure, indicating extensive reaction of NH3+ groups with the fixative. Nuclei were strongly strained by the Feulgen procedure, with no backgroud reaction. In contrast, glutaraldehyde fixation resulted in a significant background reaction in the cytoplasm and nuclei in controls for the Schiff-based stains. DMS-fixed liver stained intensely for glycogen with the Periodic acid-Schiff procedure, and biochemical analysis of glycogen retention and extractability indicated that DMS retained considerably more glycogen in sections than glutaraldehyde. DMS-fixed liver incubated for thiamine pyrophosphatase activity revealed reaction product in ER cisternae, Goli saccules and bile canaliculi. Peroxisomes were strongly reactive for catalase activity after incubation in diaminobenzidine medium, and reaction product of glucose-6-phosphatase activity was considerably greater following DMS fixation than after glutaraldehyde. Biochemical studies revealed up to twice as musch residual activity of glucose-6-phosphatase after DMS fixation. These results suggest that DMS may be useful as a primary fixative for certain cytochemical procedures.     

Influence of two different fixatives on the identification of plasma cells in human rectal mucosa

Plasma cells in sections of bisected human rectal biopsy specimens, fixed in two alternative fixatives, were enumerated after staining by an indirect immunoperoxidase procedure intended to demonstrate immunoglobulin-containing cells. The counts of immunoperoxidase-positive plasma cells were significantly higher after fixation in formol sublimate than after fixation in formol saline. Formol sublimate appears to be a more reliable fixative than formol saline for specimens of rectal mucosa in which quantitation of plasma cells, stained for intracellular immunoglobulin by an immunoperoxidase technique, is intended.     

The binding of [3H]chlorambucil to nuclear proteins of the Yoshida ascites sarcoma.

The binding of [3H]chlorambucil to nuclear proteins, extracted from Yoshida ascites sarcoma cells at 6 h and 24 h after administration of 3H-labelled drug to tumour-bearing animals, has been examined. Both covalent and non-covalent binding was detected. Considerably more drug was found associated with the proteins isolated from the tumour sensitive to the effects of the drug compared with similar proteins isolated from the tumour with an acquired resistance to the effects of alkylating agents. The two-fold difference in binding to total cell protein is attributed to a higher intranuclear protein binding in sensitive cells. In particular the soluble nuclear sap fraction from sensitive cells bound at least five times as much drug as the corresponding fraction from resistant cells. Low levels of binding to histones were demonstrated compared with that to the non-histone chromatin proteins. Binding to the nuclear sap and non-histone chromatin proteins was principally to high molecular weight protein species; these did not appear to represent aggregation products as scans of stained polyacrylamide gels of the extracted protein fractions were unaltered by the treatment of tumour-bearing animals with chlorambucil. Binding to the nuclear proteins from sensitive cells tended to persist over a 24-h period, whereas it was considerably reduced in resistant cells.