Showing papers in "Journal of Histochemistry and Cytochemistry in 1974"

Periodate-lysine-paraformaldehyde fixative a new fixative for immunoelectron microscopy

Abstract: A new fixative which primarily stabilizes carbohydrate moieties was developed for immunoelectron microscopy. It contains periodate, lysine and paraformaldehyde. Theoretically, the carbohydrates are oxidized by periodate and cross-linked by lysine. The fixative can preserve antigenicity as well as paraformaldehyde and ultrastructure as well as glutaraldehyde. Using this fixative and the peroxidase-labeled antibody technique, basement membrane antigen was localized within the cisternae of endoplasmic reticulum of parietal yolk sac cells and in extracellular basement membranes with adequate tissue successfully accomplished by conventional

Peroxidase-labeled antibody. A new method of conjugation

Abstract: A new method of conjugating horseradish peroxidase with proteins was developed. The carbohydrate moiety of fluorodinitrobenzene-blocked peroxidase was oxidized with so- dium periodate to form aldehyde groups. The peroxidase-aldehyde was then bound to free amino groups of proteins unidirectionally at high efficiencies. Peroxidase-labeled immune- globulin retained its immunologic as well as enzymatic activities. Horseradish peroxidase (HRPO), when cou- pled to immunoglobulin G (IgG), has proven to be a useful marker for immunohistochemistry (13). In contrast to other immunohistochemical markers, such as fluorescein (7) and ferritin (20), HRPO may be used for both light and electron microscopy. It is especially suitable for the intracellular localization of antigens at the ultrastructural level since HRPO (40,000 molec- ular weight) is considerably smaller than fer- ritin (650,000 molecular weight); thus, HRPO- labeled IgG (HRPO-IgG) has superior penetra- tion properties. HRPO is usually coupled to IgG using bifunc- tional reagents. With earlier methods, the con- jugation reactions were carried out in the pres- ence of HRPO, IgG, and p,p'-difluoro-m,m - din tro-diphenyl sulfone (FNPS) (13), (1-cyclohexyl- 3-(2-morpholinoethyl)) carbodiimide metho-p- toluenesulfonate (4), cyanuric chloride (1), bis-

The histochemical demonstration of o-acylated sialic acid in gastrointestinal mucins their association with the potassium hydroxide-periodic acid-schiff effect

Abstract: O-Acylated sialic acids have been demonstrated histochemically in the epithelial mucins of the lower end of the gastrointestinal tract. The potassium hydroxide-periodic acid-Schiff effect has been ...

The hemalog d white cell differential system

Abstract: The Hemalog D system performs automated differential white cell counts utilizing principles of cytochemistry, electro-optical measurement and signal logic processing. Using 0.4 ml of anticoagulated whole blood and an automated continuous flow staining technique, the system is designed to process a new sample each minute. The decision logic classifies individual cells by size and intensity of staining as they flow through detectors designed to measure light loss and light scattering simultaneously. With this system, 10,000 cells/ sample are classified in less than 1 min. Alcian Blue identifies basophils when used in the presence of quaternary ammonium salts and other counterions. Monocytes are the only cells stained in an esterase method using α-naphthol butyrate at pH 6.1 with hexazonium pararosanilin as coupler. This unstable diazonium is produced on stream continuously from stable components. Size and peroxidase staining serve to classify the remaining cells. Lymphocytes and large mononuclear cells are ...

Ultrastructural immunocytochemical localization of lysozyme in the Paneth cells of man.

Abstract: Human lysozyme was localized immunocytochemically at the ultrastructural level within Paneth cells of man by use of the unlabeled antibody enzyme method. Specific staining for lysozyme was observed...

A more sensitive and specific histochemical peroxidase stain for the localization of cellular antigen by the enzyme-antibody conjugate method

Abstract: In an attempt to increase the sensitivity and specificity of immunohistochemical procedures using peroxidase-babeled antibody, we have modified the technique. This modification takes advantage of the pH optimum of horseradish peroxidase. The best buffer salts, hydrogen peroxide concentration, time of staining, fixation after incubation with labeled antibody and counterstains were determined empirically. Compounds similar to the substrate are known to undergo photooxidation, and specificity was enhanced by keeping the solutions in the dark.

The unlabeled antibody enzyme method of immunocytochemistry quantitative comparison of sensitivities with and without peroxidase-antiperoxidase complex

Abstract: Araldite sections of rat pituitary intermediate lobe were used with anti-17-39 adrenocorticotropin in the unlabeled antibody enzyme method to compare electron microscopic immunocytochemical staining by peroxidase-antiperoxidase complex (PAP) with antiserum or purified antibody to peroxidase followed by peroxidase (PO). Quantitation of normalized optical densities of secretory granules offered high significance comparison (P < 0.0001) of experimental with control values and of experimental values with each other. Use of purified antibody (prepared by a new density gradient method) yielded four times higher sensitivity than antiserum to PO, while a 6.5-fold increase would have been expected from the degree of contamination of anti-PO in the serum by nonanti-PO immunoglobulin. Use of PAP was four to five times more sensitive than purified anti-PO and 20 times more sensitive than antiserum to PO. The increased sensitivity of PAP is explained by the high over-all binding affinity of PO for anti-PO in the cycli...

Histochemical demonstration of (Na+-K+)-activated adenosine triphosphatase.

Abstract: Rats were perfused with a l2% solution of polyvinyl pyrrolidone (pH 7.25). Frozen sections were prepared from spinal cord and kidney and incubated under conditions that are known to be optimal for demonstrating the p-nitrophenylphosphatase activity of (Na+-K+)-adenosine triphosphatase (ATPase). After incubation, the sections were treated sequentially with cobalt chloride and ammonium sulfide to visualize the reaction product. The reaction product was localized perferentially to the neuropil of the spinal cord and to the distal tubules of the kidney. In addition to the histologic localization, the following observations indicate that the histochemical reaction is specific for demonstrating (Na+-K+)-ATPase activity: (a) the conditions that are optimal for the p-nitrophenylphosphatase activity of purified (Na+-K+)-ATPase were also optimal for our histochemical method; (b) the histochemical reaction was inhibited by ouabain; and (c) the p-nitrophenylphosphatase reaction occurred under conditions that inhibit ...

Tissue fixation with diimidoesters as an alternative to aldehydes i. comparison of cross-linking and ultrastructure obtained with dimethylsuberimidate and glutaraldehyde

Abstract: Diimidoesters are bifunctional reagents which react with the e-amino group of lysine, forming intermolecular cross-links with minimal alteration of the native properties of proteins. To determine t...

Localization of lysozyme in normal rat tissues by an immunoperoxidase method

Abstract: bridge technique has been utilized for the localization of lysozyme (LZM) in the cells and tissue of the normal rat. Specific LZM staining was found in the proximal tubules of the kidney, Paneth cells of the small intestine and alveolar macrophages. LZM staining was also demonstrated in macrophages in the perifollicular sinusoids of an activated lymph node. The immunoperoxidase method appears to have significant advantages over previously described methods for LZM localization, and it should also be adaptable to electron microscopic studies. Lysozyme (LZM) is widely hut selectively distributed in the tissues of animals. LZM’s antibacterial action is well documented (15) but it has also been postulated to have an effect on mammalian cells and their constituent membranes which may be physiologically significant (14). As discussed subsequently, several methods have been used to demonstrate LZM in cells and tissues. The purpose of this paper is to describe studies of the localization of LZM in the tissues of the normal rat using the immunoperoxidase method (12). This method appears to possess the advantages of greater sensitivity and specifIcity as compared with other procedures and is also applicable to frrmalinfixed and paraffin-embedded sect ions.

The effect of digestion with chondroitinases upon certain histochemical reactions of mucosaccharide-containing tissues.

Abstract: The effect of digestion with chondroitinases ABC and AC upon certain histochemical reactions of mucosaccharide-containing vertebrate tissues was studied in various species including man. Differing ...

Microfluorometric analysis of deoxyribonucleic acid replication kinetics and sister chromatid exchanges in human chromosomes

Abstract: Fluorescence of the dye 33258 Hoechst, when bound to chromosomes, is partially quenched by the incorporation of 5-bromodeoxyuridine into chromosomal deoxyribonucleic acid (DNA). This effect allows microfluorometric analysis of DNA synthesis. Metaphase chromosomes from cultured human leukocytes which have incorporated 5-bromodeoxyuridine for a portion of the DNA synthesis period exhibit reduced 33258 Hoechst fluorescence in 5-bromodeoxyuridine-containing regions. Regions synthesizing DNA during a particular interval can thus be highlighted by the appropriate protocol of 5-bromodeoxyuridine administration. Chromosomes from cells which have replicated twice in medium containing 5-bromodeoxyuridine exhibit one brightly and one dully fluorescing chromatid, reflecting incorporation of 5-bromodeoxyuridine into one or two chains of chromatid DNA, respectively. Sister chromatid exchanges, evident as sharply demarcated reciprocal alterations in fluorescence along chromosomes, can be located relative to quinacrine b...

Destruction of endogenous peroxidase activity in order to locate cellular antigens by peroxidase-labeled antibodies

Abstract: Convalent enzyme-antibody conjugates have been extensively employed for the immunochemical localization of cellular antigens (2. 5). This method is sometimes limited by the necessity to distinguish between endogenous enzyme activity and enzyme activity due to labeled antibody. This distinction is facilitated if the endogenous enzyme activity is destroyed while antigenic activity is preserved. Straus (6) used 1% sodium nitroferricyanide and 1% acetic acid in methanol as a fixative to destroy endogenous peroxidase activity. This fixative was recently applied in this laboratory to immunochemical localization of cellular antigens by a peroxidaseantibody conjugate. Streefkerk (7) destroyed endogenous peroxidase activity by treatment with methanol followed by hydrogen peroxide. Since methanol may destroy some cellular antigens, a different, methanolfree, fixing solution was sought. Figure 1 shows a 10-s frozen section of rabbit spleen which was fixed in absolute ethanol for 15 mm at room temperature. The section was incubated in a humidified chamber under a drop of 0.85% sodium chloride in 0.01 M sodium phosphate buffer, pH 6.8 (PBS), for 3 hr. The slide was then stained for peroxidase for 5 mm using a method similar to Graham and Karnovsky (3) as modified by Mazurkiewicz and Nakane (4). This slide shows the endogenous peroxidase activity of the rabbit spleen used. Figure 2 is the next serial section of the spleen. This section was fixed with 0.074% hydrochloric acid in ethanol (0.2 ml concentrated hydrochloric acid in 100 ml ethanol) for 15 mm at room temperature and incubated with PBS and stained as above. The endogenous peroxidase activity is abolished under these conditions. Figure 3 is the next serial section of the spleen. This section was fixed with 0.074% hydrochloric acid in ethanol for 15 mm at room temperature. The section was then incubated for 3 hr under a drop of PBS containing sheep antirabbit -y-globulin -peroxidase conjugate (0.50 mg protein/ml) prepared by a method similar to Avrameas (2). The section was washed twice for 1 mm and once for 30 mm in PBS and stained for peroxidase. In some instances, after immunohistochemical antigen location by peroxidase, the sections were counterstained with methyl greenpyronin (1) (Edward Gurr, Ltd., Michrome Labs, Bucks, England). The methyl green-pyronin did not affect the localization of the peroxidase reaction product and demonstrated that the cells which stained immunohistochemically were plasma cells. The antigenicity of the plasma cell -y-globulins was not destroyed by this fixation. By experiments similar to the above, plasma cells were also specifically stained in sections of rabbit appendix and lymph node using antirabbit ‘y-globulin prepared from both sheep and goat immune sera. Plasma cells were not stained by rabbit antiferritinperoxidase conjugates. The sections were fixed with 0.074% hydrochloric acid in ethanol as described. In these tissues, the endogenous peroxidase activity was destroyed while the antigenic activity remained intact. All slides were made in duplicate. In summary, new conditions for fixation are described which destroy endogenous peroxidase activity but not antigenic activity of rabbit -y-globulins. Hopefully, these conditions will be useful for other antigens as well.

Cell cycle analysis from computer synthesis of deoxyribonucleic acid histograms

Abstract: A method is presented for the computer synthesis of deoxyribonucleic acid (DNA) histograms, based on estimates of cell distribution around a life cycle, DNA synthesis rate and instrumentally introduced dispersion. Synthetic histograms are compared with experimental DNA histograms for two cell systems; in one, the cell population is in asynchronous exponential growth (steady state), in the other, the cell population is recovering from a block at the GJS phase interface (nonsteady state). The average phase transit times and the associated dispersions are determined, using a DNA histogram synthesis technique, for the nonsteady state population. The quantitation of cell populations on the basis of the frequency distribution of cellular deoxyribonucleic acid (DNA) is becoming increasingly common as high speed cytophotometric systems (3, 4, 7, 11, 13, 18) become available. It is therefore important that such DNA frequency distributions (DNA histograms) be well understood. A computer model of cell proliferation is presented in this report-a model which can be used to synthesize DNA histograms. The model is based on reasonable assumptions about kinetic parameters (9), rate of DNA synthesis (19), cell distribution around a life cycle (5, 6, 12, 14-16) and instrumentally introduced dispersion . This modeling concept not only provides a basis for a more complete understanding of how these parameters affect experimentally determined DNA histograms but also provides a new method for extracting cell cycle parameters of perturbed populations. The report first illustrates how a DNA histogram can be synthesized for the steady state situation of a population in asynchronous, exponential growth (steady state implies that the cell distribution is time-invariant). After the synthesis technique is illustrated for this steady state population, the time invariance restriction is relaxed and nonsteady state conditions such as those found in synchronous cell populations are discussed. Once the modeling technique is illustrated, 1 This work was performed under the auspices of

Computer-controlled multiparameter analysis and sorting of cells and particles.

Abstract: A new high speed instrument for the multiparameter analysis and separation of cells and subcellular particles under computer control has been constructed. It incorporates several new features, the combination of which is believed to make the system uniquely capable of attacking a wide variety of biologic questions. In this paper we present the biologic objectives as well as the salient new features of the instrument.

Localization of carbonic anhydrase in avian gastric mucosa, shell gland and bone by immunohistochemistry.

Abstract: chromatography. The anti-CA immuno-’y-globulins were tested by immunoelectrophoresis and judged to be highly specific for CA. Fluorescein isothiocyanate-conjugated goat antirabbit v-globulins were used in the indirect fluorescent antibody localization of CA in cryostat sections. Specific fluorescence of CA was observed in gastric mucosal lining cells, shell gland columnar and tubular gland cells, red blood cells, erythroblasts and osteoclasts. Specific fluorescence was absent in the several immunologic controls, including the blocking test. Specific fluorescence was also lacking in tissue constituents which contain no detectable amounts of CA, i.e., muscle, connective tissue, blood vessels and nuclei. A significant finding in this study is the occurrence of cross-reactions between antibodies to red blood cell CA and CA from other tissues, indicating the immunologic simi!arity of CA from different tissue sources. Identification of cells that contain carbonic anhydrase (CA), EC 4.2. 1. 1, is of considerable importance in understanding both cell and organ function. The role of CA is thought to be associated with the protolysis of water which -. +

Problems of specificity in the use of a strontium capture technique for the cytochemical localization of ouabain-sensitive, potassium-dependent phosphatase in mammalian renal tubules.

Abstract: The p-nitrophenyl phosphate-strontium procedure for the localization of the phosphatase component of Na-K-activated adenosine triphosphatase was evaluated using rat renal cortex as a test tissue. The results obtained by light microscopy were unexpected in that reaction product was found only on the brush borders of proximal tubule cells; this reaction was ouabain-resistant, K-independent and partially Mg-dependent, but could be completely inhibited by l-tetramisole. Electron microscopy showed that a reaction was also present on the cytoplasmic surfaces of the lateral and basal plasma membranes of the proximal and distal tubule cells. That seen in the distal tubule was sensitive to ouabain but not to l-tetramisole, whereas that in the proximal tubule showed a mixture of ouabain-sensitive and l-tetramisole-sensitive components. It is concluded that the procedure as originally described is not specific, demonstrating alkaline phosphatase as well as Na-K-adenosine triphosphatase, but that this problem may be overcome by the use of an alkaline phosphatase inhibitor.

Automated analysis of deoxyribonucleic acid, protein and nuclear to cytoplasmic relationships in tumor cells and gynecologic specimens

Abstract: Quantitative two-color fluorescence staining techniques, coupled with flow system multiparameter cell analysis and sorting instrumentation, have been used for rapid, simultaneous determination of deoxyribonucleic acid, protein, nuclear (N) and cytoplasmic (C) diameters and N : C ratios in mammalian tumor cells and human gynecologic specimens. Cells stained in suspension for deoxyribonucleic acid and total protein content, respectively, with propidium iodide (red fluorescence) and fluorescein isothiocyanate (green fluorescence), enter a flow chamber and intersect an argon laser beam which excites cellular fluorescence. Optical sensors measure both red and green fluorescence plus the time duration of each fluorescence signal which is proportional to nuclear and cytoplasmic diameters, respectively. The resulting signals are processed and displayed as frequency distribution histograms using a multichannel pulse height analyzer. Cells are also sorted based on N : C ratios. Illustrative examples of preliminary two-color fluorescence analysis and sorting of mouse squamous tumor cells and human exfoliative vaginal cells are presented. The development of high speed, flow system cell sorting techniques based on physical and biochemical measurements on single cells has provided a new dimension to biologic investigation by being able to isolate physically cells having particular properties from heterogeneous cell populations (6, 8, 10). Such systems have been used to sort human leukocytes based on volume measurement (15), to isolate antigenbinding cells which are precursors to antibodyproducing cells by immunofluorescence (9) and to separate cells based on ultraviolet absorption and visible light scatter (10). Instrumentation for analyzing and sorting cells according to simultaneous measurement of cell volume, total or two-color fluorescence and light scatter has been developed recently ( 13), incorporating 5everal preexisting analytical techniques used in flow system analysis (2, 11, 16). This system has recently been used to sort acridine orangestained human leukocytes based on cytoplasmic granulation (14), model mouse tumor cells according to deoxyribonucleic acid (DNA) content

Observations implicating an extracellular enzymic mechanism of control of bone morphogenesis

Abstract: Data on physicochemical conditions leading to loss of the bone morphogenetic property of bone matrix in neutral buffer solutions support the concept of an enzymic control mechanism better than a ch...

Automated classification of normal and abnormal leukocytes.

Abstract: The development of an automated system for counting and classifying normal and abnormal leukocytes in peripheral blood smears is described. General requirements are discussed and the results of a simulation experiment are presented. A sample of 1572 leukocytes, divided equally among 17 types, was photographed and analyzed using computerized pattern recognition techniques. Various geometrical, color and texture parameters were extracted from the cell images and an optimal set of 20 were used in several computerized classification runs. Training on one-half of the sample and classifying the other half resulted in an over-all correct classification of between 67 and 77% depending on the definition of classification error. When only normal cells are considered, correct classification is obtained for 9l.5% of the cells.

Electron probe microanalysis of skeletal muscle.

Abstract: to chelate calcium and with Tris chloride to replace sodium. Osmium-fixed controls were also analyzed. Both crystal-diffracting and energy-dispersive analyzers were employed, the former for accurate quantitation of calcium content and the latter for qualitative assessment of other elements present. Significant concentrations of calcium occurred in the dense parts of the nucleus, the triads, the thin filaments and the sarcolemma. The amount of calcium was greatly decreased by ethyleneglycoltetraacetate pretreatment and slightly increased by sodium removal. After osmium fixation alone, little calcium was detected with no localization.

Sensitivity in electron microscope autoradiography for 125i

Abstract: Sensitivity in electron microscope autoradiography was determined for 125I. Values are given using Ilford L4 and Kodak NTE emulsions combined with different developers. The extent of self-absorption as a function of section thickness and heavy metal staining and the effect of radiation dose (dose dependence) were assessed. It was found that the over-all efficiency for 125I was better than that for tritium and that, as is the case with tritium, there is a distinct "dose dependence" especially when Microdol X is the developer. Self-absorption studies indicate that self-absorption is increased by about 15% when the specimen thickness is increased from 300 to 1000 A. An increase of under 15% is also introduced by heavy metal staining of sections in this thickness range.

Cytochemical localization of brain nicotinamide adenine dinucleotide phosphate (oxidized)-dependent dehydrogenases qualitative and quantitative distributions

Abstract: This study compares the histochemical and microchemical localizations of nicotinamide adenine dinucleotide phosphate (reduced) and nicotinamide adenine dinucleotide (reduced) diaphorases and four nicotinamide adenine dinucleotide phosphate (oxidized)-dependent enzymes (glucose 6-phosphate, 6-phosphogluconate, malate and isocitrate dehydrogenases) in areas of rat metencephalon and spinal cord. For the four nicotinamide adenine dinucleotide phosphate (NADP) enzymes, the pattern of localization following use of a modified tetrazolium procedure was compared with quantitative data obtained by microdissection from the same areas in adjacent sections. Optimal experimental conditions for reaction pH, temperature, substrate, cofactor and divalent cation concentrations were used for both the quantitative analysis following microdissection and the histochemical tetrazolium procedure. Consecutive sections were also examined for isocitrate dehydrogenase (nicotinamide adenine dinucleotide (oxidized)) and nicotinamide a...

A microspectrofluorometer with epi-illumination operated under computer control.

Abstract: Until recently the recording of fully corrected excitation and fluorescence spectra from fluorophores in microscopic preparations has been a time-consuming procedure. A new type of microspectrofluorometer with epi-illumination has been developed. This instrument is operated on line with a digital computer permitting automatic operation of monochromator motors, electrical shutters and correction of spectra. The electrical shutters are used for a rapid change between low intensity dia- and strong epi-illumination. Exposure to epi-illumination is limited to the short period of recording the spectra. A new optical system permits centering of structures to be measured, using the binocular head for viewing the illuminated image of the monochromator slit and the fluorescent image excited with dia-illumination simultaneously. New objectives and eyepieces with a high light collecting power permit the visualization of weak fluorescence obtained with low intensity dia-illumination.

Determination of the polarization optical properties of the amyloid-Congo red complex by phase modulation microspectrophotometry.

Abstract: The polarization properties responsible for the classical “green birefringence” of the amyloid-Congo red complex have been determined by a new optical method, phase modulation microspectrophotometry. This method now makes possible the measurement of one optical property at a time (birefringence, optical rotation, linear dichroism and circular dichroism throughout the visible spectrum) in complex specimens in which visible contrast in polarized light is the result of a mixture of polarization effects. The green birefringence is explained by a combination of optical effects, the strongest of which are dispersion of birefringence and linear dichroism superimposed on the smaller effects of circular dichroism and optical rotatory dispersion. The interaction of the planar dye molecules with the amyloid protein induces an extrinsic Cotton effect. Amyloidosis is a disease process in which fibrillar material is deposited in the intercellular spaces of various organs and tissues. Two main varieties of amyloidosis have recently

Electrophoresis of elastase-like esterases from human neutrophils

Abstract: been demonstrated after separation by cationic disc gel electrophoresis. Two substrates, l-naphthyl N-acetyl-DL-alanine (NAcAla) and l-naphthyl butyrate, were used routinely in a simultaneous coupling azo dye method to demonstrate the esterases. Cytochemically, the neutrophil granules stain very poorly with a-unsubstituted carboxylic acid esters such as 1-naphthyl butyrate or 1-naphthyl acetate but stain selectively and intensely at pH 7.0 with two classes of naphthol esters; a-halo-substituted esters such as l-naphthyl 2-bromobutyrate or naphthol AS-D chioroacetate or a-amino-substituted carboxylic acid esters such as l-naphthyl N-acetyl-m�-alanine or l-naphthyl N-acetyl-u-alanyl-t�-alanyl-i�-alanine. Cationic zymograms showed three major neutrophil esterases which hydrolyze NAcAIa and l-naphthyl N-acetyl-L-alanyl-L-alanyl-L-alanine vigorously and l-naphthyl butyrate very poorly, which provided us with the first evidence that these enzymes might be proteolytic. Cationic zymograms of a mixture of purified enzymes (reference enzymes), pancreatic elastase, chymotrypsin and trypsin, were compared to zymograms of neutrophil extracts after assaying with NAcAla. The neutrophil esterases and elastase hydrolyzed NAcA1a vigorously; chymotrypsin and trypsin hydrolyzed it moderately. More precise characterization of the neutrophil esterases was obtained with chloromethyl ketone inhibitors which have the advantage of being irreversible and also can be synthesized with a substrate-like moiety which confers specificity. Neutrophil extracts and the mixture of reference enzymes were preincubated before electrophoresis with either N-acetyl-L-alanyl-L-alanyl-L-alanine chloromethyl ketone - �‘ -. - �

Deoxyribonucleic acid cytochemistry for automated cytology

Abstract: Fluorescent staining for intracellular deoxyribonucleic acid content is being investigated as a means of making automated measurements quantitative and reliable. Ambiguities regarding the role of sulfur dioxide in Feulgen staining chemistry have been resolved showing that: (a) SO2 does not covalently bind to the anilinium moieties of pararosaniline; (b) SO2 does promote the binding of pararosaniline within the nuclei of hydrolyzed cells; (c) SO2 also does not bind to dyes such as acriflavine used in fluorescent-"Feulgen" staining and (d) SO2 is not required for strong, nucleus-specific acriflavine staining of nuclei of hydrolyzed cells. These findings permit simplifications of the usual fluorescent-"Feulgen" procedures. Attempts to use fluorescent intercalating dyes as vital stains for deoxyribonucleic acid content have been unsuccessful.

Simultaneous differential staining by a cationic carbocyanine dye of nucleic acids, proteins and conjugated proteins ii. carbohydrate and sulfated carbohydrate-containing proteins

Abstract: The cationic carbocyanine dye l-ethyl-2-[3-(l-ethylnaphtho[l,2d]thiazolin-2-ylidene)-2-methylprolpenyl]-naphtho[1, 2d]thiazolium bromide stains several classes of macromolecules differentially in histologic sections. Most proteins are red at pH 4.3 and pink or unstained at pH 2.8. The caseins are blue at pH 4.3 and unstained at pH 2.8. Nuclei stain purple, mast cells stain red-purple, cartilage stains purple and mucoproteins stain blue-green at both hydrogen ion concentrations. The nature of some of the macromolecules involved in these color reactions has been determined by the use of chemical and enzymatic procedures and by staining films of glycosaminoglycuronoglycans, proteins and conjugated proteins. The method requires less than 1 hr staining time for most tissues; the stain is stable when kept in the dark and has the advantage of distinguishing several macromolecules in tissues simultaneously. The simplicity of the method and the ability to discriminate classes of macromolecules in tissues should ma...