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

Summary 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.Dedicated to Prof. Dr. E. Schauenstein on the occasion of his 70th birthday     

Quantitative microspectrophotometrical determination of protein thiols and disulfides with 2,2'-dihydroxy-6,6'-dinaphthyldisulfide (DDD). The variety of DDD-staining methods demonstrated on Ehrlich ascites tumor cells

2,2'-dihydroxy-6,6'-dinaphthyldisulfide (DDD) reacts with both protein thiol groups and with protein disulfides (N?hammer 1977). By varying the pH of the DDD-reaction, as well as the reaction times, the complex reaction became specific with respect to the histochemical demonstration of protein-SH groups. Furthermore, the application of the histochemical DDD-reaction following quantitative blockade of the protein-SH groups enabled the demonstration of distinctive DDD-reactive disulfides. The specificity and the extent of the different histochemical DDD-staining methods were investigated by comparing macroscopically determined values of the protein-SH-contents, and the contents of the different kinds of disulfides in Ehrlich-ascites-tumor cells (EATC) (Modig 1968; Hofer 1975), with microspectrometrical values determined with the MCN-method of N?hammer et al. (1981), and with microspectrometrical values measured on EATC after staining with the modified DDD-methods. Also, the method for the histochemical demonstration of protein-SH with DDD after the reduction of the disulfides with thioglycolate was investigated and conditions were found by which the protein-SH content could be determined quantitatively with DDD and Fast blue B after the reduction of the disulfides. With the aid of the MCN-method (N?hammer et al. 1981), the intracellular disulfide interchange reaction was investigated, leading to pH-dependent changes of the SH-SS-ratio of fixed cells during their incubation in aqueous media. In addition the possibility of protein loss during the long incubation times of the fixed cells in the DDD-solutions was investigated. For the quantitative microscpecrometrical determination of the protein content of EATC the so-called tetrazonium-coupling method, optimized by N?hammer (1978) and calibrated by N?hammer et al. (1981), was used.     

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 improved acriflavine-Feulgen method.

The acriflavine-Feulgen method for the histochemical demonstration of deoxyribonucleic acid was modified by staining hydrolyzed cells with 0.01% acriflavine dissolved in 90% ethanol. This method offered the following advantages: (a) it simplified the preparation of the acriflavine-Feulgen reagent; (b) it left the cytoplasm essentially unstained while staining the nuclei bright green in hydrolyzed cells and left the cytoplasm and nuclei essentially unstained in unhydrolyzed cells; (c) it eliminated poorly defined reagents from the staining solutions. Because of these staining properties, this technique may be especially useful in the quantitative determination of deoxyribonucleic acid by cytofluorometry.     

Evaluation of immunocytochemical detection methods of incorporated bromodeoxyuridine in hybridomas

Bivariate distributions obtained from nominal acid hydrolysis or thermal treatment methods used in the cell cycle analysis of incorporated bromodeoxyuridine were shown to be unacceptable with hybridomas. Four different cell treatment and staining methods were compared. These methods are acid hydrolysis, thermal denaturation, nuclei extraction with pepsin digestion, and simultaneous pepsin digestion and acid hydrolysis. The nuclei extraction method was determined to be the most appropriate for the immunocytochemical staining of incorporated bromodeoxyuridine in hybridomas. The resulting bivariate distribution provides a clear distinction between labelled and unlabelled cell fractions. The method based on nuclei extraction with pepsin digestion was optimized for a hybridoma line used in this study.     

A highly sensitive method for the histochemical demonstration of copper in normal rat tissues

Earlier, widely used histochemical methods for the demonstration of copper are capable of detecting only extremely high tissue levels of this metal (generally only in pathological states, e.g. Wilson's disease, or in cases of copper intoxication), because of their low sensitivity. The specificity of these methods has also proved to be unsatisfactory. We present a new method based on the release of bound (unreactive) copper by trichloroacetic acid, its primary precipitation using magnesium dithizonate, and intensification of the staining (secondary precipitation) using silver nitrate. Using this method, copper is demonstrable in various tissues of normal rats (brain, stomach, liver, small intestine, spleen, pancreas, kidneys) in the form of reddish to pink staining. This method can also be applied to locate pathologically high levels of copper.     

Quantification of the histochemical staining for carbonyles and DNA using 3-hydroxy-2-naphthoic acid hydrazide and fast blue B

Fixed cells and tissues pretreated with 4-hydroxynonenal were used as models for the histochemical demonstration of protein bound aldehydic groups. The aldehydes were stained with both a modification of the 2,4-dinitrophenylhydrazine method (2,4-DNPH) and the optimized staining using 3-hydroxy-2-naphthoic acid hydrazide and Fast blue B (NAH-FB). A correlation has been found between the specific microphotometric mean integrated maximum absorbance values of cells and tissues stained with 2,4-DNPH and with NAH-FB (cc = 0.999). The maximum absorbance measured after 2,4-DNPH-staining (epsilon 367 = 21,000) were 1.893 +/- 0.072 (P less than 0.01) times that of NAH-FB-staining at 550 nm. Microphotometrically determined DNA-values of different cells stained with the NAH-FB-DNA-method correlated with those determined with methods of analytical biochemistry and published by other authors.     

The demonstration of acid alpha-naphthyl acetate esterase activity in human lymphocytes: usefulness as a T-cell marker.

The histochemical demonstration of nonspecific acid α-naphthyl acetate esterase (ANAE) activity was evaluated as a T lymphocyte marker primarily with the sheep erythrocyte (E) assay. A distinctive staining pattern characterized T lymphocytes which could be readily distinguished from monocyte staining. The percentage of E⁺ and ANAE⁺ lymphocytes was nearly always comparable in the peripheral blood and lymphoid tissue from normal and selected patients, including those with acute and chronic lymphocytic leukemia. Divergences were noted in certain other tissues including spleen and thymus. Certain mitogen-stimulated cells lost their ANAE activity while retaining their ability to form e_n rosettes. Atypical and variable staining patterns were observed in established lymphoid cell lines. The histochemical demonstration of ANAE is simple and reproducible; preparations may be counterstained for cytomorphologic detail and mounted as a permanent record. Certain disadvantages are discussed. The method represents a practical alternative to E rosette assays. It is particularly well suited for certain routine laboratories.     

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.     

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.     

Histochemical demonstration of phospholipase B (lysolecithinase) activity in rat tissues.

A method has been developed for the histochemical demonstration of phospholipase B (lysolecithinase) of rat tissues. The enzyme attacks lysolecithin with liberation of 1 mole of glycerylphosphorylcholine and 1 mole of fatty acid. The recommended procedure involves use of 6-10 micro frozen sections, fixed in cold calcium-formol and incubated at 37 degrees C in Tris buffered medium at pH 6.6 containing 2.2 X 10(-3) M lysolecithin and 1% cobalt acetate. The fatty acid liberated by enzymatic hydrolysis is trapped as a cobalt precipitate and is then converted to a black-brown precipitate by treatment with dilute ammonium sulfide in cold isotonic saline. Equivalent amounts of fatty acid and glycerylphosphorylcholine are recovered by extraction and analysis of the incubated sections and of the incubation medium, thus proving that lysolecithin hydrolysis occurs under the proposed reaction conditions. Staining is reduced by treating the sections with copper ions, mercury compounds, alcohols, acetone and by heating at 60 degrees C prior to incubation with substrate. Lowering of the pH of the incubation medium has similar effect. These findings are interpreted as evidence of the enzymatic nature of the reaction. Cells exhibiting a positive staining are found in the lamina propria of the intestinal villi and crypts, in the red pulp of the spleen and in the interstitial tissue of lung, liver and thymus. Similar elements are present in bone marrow smears and in leukocyte preparations obtained by peritoneal lavage. The morphologic and staining characteristics of these cells correspond to those of the eosinophilic leukocytes. Physical and chemical agents (x-irradiation, corticosteroids) which sharply decrease the number of eosinophils also reduce the number of cells shown histochemically to hydrolyze lysolecithin. A correspondent diminution of phospholipase B activity of homogenates of the same tissues can be shown in vitro. Differences in tissue distribution and chemical properties distinguish the phospholipase B from less specific esterases and lipases.     

Potential inhibitors of rat tooth alkaline phosphatase studied by means of different histochemical techniques.

Two histochemical methods for demonstration of alkaline phosphatase activity, a lead pyrophosphate- anda naphtholphosphate technique, were compared. Since different results may be due to methodological differences as well as different enzyme activities, the enzymatic hydrolysis of the naphtholphosphate was visualized both by means of an azo-dye coupler and by lead-capturing of the liberated phosphate ion. Various potential inhibitors of alkaline phosphatase activity (diphosphonate, D-penicillamine, and sodium fluoride) were also tested. The use of diphosphonate and D-penicillamine resulted in inhibited or reduced staining, which could mainly be explained by an interference by these compounds with components in the incubation media rather than with the enzyme itself. The addition of sodium fluoride had no effect on the naphtholphosphate staining pattern irrespective of capturing method, whereas the odontoblastic pyrophosphate splitting alkaline phosphatase appeared to be sensitive to sodium fluoride, suggesting the presence of two alkaline phosphatases in odontoblasts.     

Quantitative microspectrophotometrical determination of protein thiols and disulfides with 2,2′-dihydroxy-6,6′-dinaphthyldisulfide (DDD)

Summary 2,2-dihydroxy-6,6-dinaphthyldisulfide (DDD) reacts with both protein thiol groups and with protein disulfides (Nöhammer 1977). By varying the pH of the DDD-reaction, as well as the reaction times, the complex reaction became specific with respect to the histochemical demonstration of protein-SH groups. Furthermore, the application of the histochemical DDD-reaction following quantitative blockade of the protein-SH groups enabled the demonstration of distinctive DDD-reactive disulfides. The specifity and the extent of the different histochemical DDD-staining methods were investigated by comparing macroscopically determined values of the protein-SH-contents, and the contents of the different kinds of disulfides in Ehrlich-ascites-tumor cells (EATC) (Modig 1968; Hofer 1975), with microspectrometrical values determined with the MCN-method of Nöhammer et al. (1981), and with microspectrometrical values measured on EATC after staining with the modified DDD-methods. Also, the method for the histochemical demonstration of protein-SH with DDD after the reduction of the disulfides with thioglycolate was investigated and conditions were found by which the protein-SH content could be determined quantitatively with DDD and Fast blue B after the reduction of the disulfides. With the aid of the MCN-method (Nöhammer et al. 1981), the intracellular disulfide interchange reaction was investigated, leading to pH-dependent changes of the SH-SS-ratio of fixed cells during their incubation in aqueous media. In addition the possibility of protein loss during the long incubation times of the fixed cells in the DDD-solutions was investigated. For the quantitative microscpecrometrical determination of the protein content of EATC the so-called tetrazonium-coupling method, optimized by Nöhmmer (1978) and calibrated by Nöhammer et al. (1981), was used.Dedicated to Prof. Dr. E. Ziegler on the occasion of his 70^th birthday     

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 Hazard of Acid Differentiation in Gomori's Method for Acid Phosphatase

In his original method for the histochemical demonstration of acid phosphatase, Gomori prescribed differentiation of incubated sections by rinsing them in 14% aqueous acetic acid, to remove the nonspecifically precipitated lead deposits. According to him, the enzymatically produced lead phosphate is not washed out by this procedure. As a result of recent improvements in tissue preparation and shorter incubation time, this staining reaction as it is used now is quite sensitive to an acetic acid wash. If this wash is used as recommended originally, it may completely abolish a truly positive reaction. To avoid falsely negative results, and to compare sections of normal and pathological tissue, omission of this differentiation by acetic acid is essential. The risk of mistaking nonspecific lead precipitates in the interpretation of a positive reaction is very small, and can be avoided by running a negative control slide in which no lead phosphate can be produced enzymatically.     

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.     

Different instability of nuclear DNA at acid hydrolysis in cancerous and noncancerous cells as revealed by fluorescent staining with acridine orange

Ehrlich cancer cells and inflammatory cells in mouse ascitic fluid were hydrolyzed and stained with acridine orange (AO). The AO hydrolysis curves for G1/G2 + M phase cancer cells and inflammatory cells were differentially determined using flow cytometry by monitoring the metachromatic red-shifted fluorescence of the fluorochrome bound to the single-stranded DNA produced by acid hydrolysis. By computer fitting of the Bateman function to the hydrolysis curves, the kinetic parameters k1 (rate constant for the production of single-stranded DNA), k2 (rate constant for the degradation of the produced single-stranded DNA), and y0 (theoretical value of the single-stranded DNA present initially) were determined. It was found that the k2 value, which reflects the degree of DNA instability, was much higher for cancer cells in both the G1 and G2 + M phases than for inflammatory cells. This finding led us to develop a method for the differential AO staining of cancer cells and non-cancerous cells utilizing the different degree of DNA instability at acid hydrolysis. AO staining after hydrolysis with 2N HCl at 30 degrees C for 8.5 min was found to be the optimal method. In the 60 cases of human malignant epithelial and nonepithelial tumors tested, all of the malignant tumor cells emitted metachromatic red fluorescence, while all of the nonmalignant tumor cells (5 cases of benign tumor) and normal cells emitted orthochromatic green fluorescence when observed with a violet excitation light under a fluorescence microscope. This new technique can be a useful tool for the screening of malignancy in exfoliative cytology and also for basic cancer research.     

Application and comparison of silver intensification methods for the diaminobenzidine and diaminobenzidine-nickel endproduct of the peroxidation reaction in immunohistochemistry and in situ hybridization.

Silver-intensification methods described in the literature for the diaminobenzidine (DAB) and diaminobenzidine-nickel (DAB/Ni) endproduct of the peroxidase reaction were compared in model systems after immunoperoxidase and in situ hybridization. First, these methods were compared in immunohistochemical model systems, using the demonstration of glial fibrillar acidic protein (GFAP) and prostate-specific antigen (PSA) in paraffin sections of human brain and prostate tissue, respectively. When DAB without Ni was used as substrate, tissue argyrophilia caused considerable background staining. Only when this tissue reactivity was quenched with, e.g., CuSO4 with H2O2 or thioglycolic acid, were the results acceptable. A considerable improvement in the signal-to-noise ratio could be obtained when nickel was included in the substrate mixture. The methods that proved to be best for demonstration of GFAP and PSA made use of acid developer solutions. Subsequently, these methods were compared with other sensitive immunostaining methods for demonstration of the gamma-delta T-cell receptor in frozen lymphoid tissue. In this model a considerable increase in the number of positive cells could be obtained using silver intensification. The different methods using DAB/Ni were also compared for use in DNA in situ hybridization (DISH). In this case two model systems were used: human papilloma virus type 11 (HPV-11) DNA in condyloma tissue (abundant target model) and Epstein-Barr virus (EBV) DNA in a mononucleosis lymph node (low target model). For demonstration of HPV-11, all methods gave more or less satisfactory results, which were best with the acid developer solutions. Moreover, for demonstration of EBV DNA, a signal could be obtained only with these developer solutions. Such a method also proved suitable in double immuno-hybrido stainings for the demonstration of EBV DNA in specific antigen-positive Reed-Sternberg cells in paraffin sections of Hodgkin lymph nodes.     

Quantification of the histochemical staining for carbonyles and DNA using 3-hydroxy-2-naphthoic acid hydrazide and Fast blue B

Summary Fixed cells and tissues pretreated with 4-hydroxynonenal were used as models for the histochemical demonstration of protein bound aldehydic groups. The aldehydes were stained with both a modification of the 2,4-dinitrophenylhydrazine method (2,4-DNPH) and the optimized staining using 3-hydroxy-2-naphthoic acid hydrazide and Fast blue B (NAH-FB). A correlation has been found between the specific microphotometric mean integrated maximum absorbance values of cells and tissues stained with 2,4-DNPH and with NAH-FB (cc=0.999). The maximum absorbance measured after 2,4-DNPH-staining (₃₆₇=21 000) were 1.893±0.072 (P<0.01) times that of NAH-FB-staining at 550 nm. Microphotometrically determined DNA-values of different cells stained with the NAH-FB-DNA-method correlated with those determined with methods of analytical biochemistry and published by other authors.