Showing papers in "Histochemical Journal in 1983"

Antibodies to intermediate filament proteins in the immunohistochemical identification of human tumours: an overview.

Abstract: Intermediate-sized filament proteins (IFP) are tissue specific in that antibodies to keratin, vimentin, desmin, glial fibrillary acidic protein (GFAP) and the neurofilament proteins can distinguish between cells of epithelial and mesenchymal origin as well as of myogenic and neural origin respectively. Malignant cells retain their tissue-specific IFP, which makes it possible to use these antibodies in tumour diagnosis. Carcinomas are exclusively detected by antibodies to keratin. Monoclonal antibodies to keratin have allowed the differentiation between subgroups of epithelial tumours until now between adenocarcinomas and squamous cell carcinomas. Lymphomas, melanomas and several soft tissue tumours are distinctly recognized by antibodies to vimentin. On the other hand, rhabdomyosarcomas and leiomyosarcomas are positive for desmin, while astrocytomas give a strong reaction with GFAP antibodies. Thus, antibodies to IFP are useful tools for differential diagnosis in surgical pathology.

A simple post-embedding system for the rapid demonstration of tissue antigens under the electron microscope.

Abstract: A simple and versatile technique for the preparation of ultra-thin sections, which can be stained immunohistochemically directly on electron microscope grids, is presented. An anti-hapten immunoperoxidase procedure has been adapted for use on tissue fixed in a purified monomeric glutaraldehyde--picric acid mixture, and embedded in 'L R White', a recently formulated plastic resin. This plastic tolerates the use of partial dehydration of tissue, resulting in higher antigenic yields. In addition, no etching of ultra-thin sections is necessary, and the whole immunostaining procedure can be completed in less than 2 h. A comparison of commonly used fixatives is discussed. High-resolution micrographs showing general staining (uranyl acetate--lead citrate) of rat pancreas, and immunostaining of insulin and TSH in storage granules in perfusion-fixed rat tissue and of lambda-chain immunoreactive cells in immersion-fixed human tonsil are included as examples.

Light microscopic detection of sugar residues in glycoconjugates of salivary glands and the pancreas with lectin-horseradish peroxidase conjugates. I. Mouse.

Abstract: Salivary glands and pancreases from male rats were stained with a battery of ten different lectin-horseradish peroxidase conjugates. Qualitative and quantitative differences were observed in the content of terminal sugar residues in stored secretory glycoproteins in parenchymal cells of glands having a similar histological structure. Heterogeneity in the content of secretory glycoconjugates was also found between cells in the same exocrine glands, which were previously thought to be identical on the basis of classical morphological and histochemical staining studies. Similar differences were observed in the structure of glycoconjugates associated with the apical surface of epithelial cells lining glandular excretory ducts. Intercalated ducts presented a gland specific staining pattern different from that of the glandular secretory cell population, whereas striated duct and interlobular duct epithelial cells stained similarly in all major rat exocrine glands. A comparison of lectin binding patterns in identical histological sites in the mouse, reported in a companion paper, is provided, and the similarities and differences between these two rodent species are discussed. In addition to providing valuable information concerning the localization and structure of tissue complex carbohydrates, a comparison of staining in the same tissue sites with labelled lectins reported biochemically to have similar binding specificity has revealed interesting differences in the binding specificity of these macromolecules.

Mucosal mast cells of the rat intestine: a re-evaluation of fixation and staining properties, with special reference to protein blocking and solubility of the granular glycosaminoglycan

Abstract: Mucosal mast cells of the gastrointestinal tract constitute a separate cell line within the mast cell system of the rat, differing in several respects from the classical connective tissue mast cells and, unlike the latter, requiring special fixation techniques for their demonstration. We have examined some histochemical properties of mucosal mast cells of the duodenum and compared them with connective tissue mast cells of the tongue or skin. The results indicate that the structural integrity of the granules of both types of mast cell is partly dependent on ionic linkages between glycosaminoglycan and protein. The so far unidentified glycosaminoglycan of mucosal mast cells appears to be more soluble than the heparin of connective tissue mast cells. The strongly fluorescent binding of Berberine to the granules of connective tissue mast cells and, depending on their content, of heparin is absent from mucosal mast cells, confirming previous findings which suggested that they contain a glycosaminoglycan with a lower degree of sulphation. Aldehyde fixation by routine procedures reversibly blocks the cationic dye binding of mucosal mast cell granules. The dye binding groups may be unmasked by trypsination or by long staining times of the order of several days. The results suggest that the blocking of staining by aldehydes is caused by a diffusion barrier of a protein nature. Mucosal and connective tissue mast cells thus differ with respect to the spatial arrangement of glycosaminoglycan and protein in their granules. As a result of the study a modified method for the demonstration of mucosal mast cells in tissue sections is described, based on normal formaldehyde fixation and staining in Toluidine Blue for a long time. It has some advantages over previous methods and preserves the structure of mucosal and connective tissue mast cells equally well.

Distribution of different fibre types in human skeletal muscles. I. Method for the preparation and analysis of cross-sections of whole tibialis anterior

Abstract: The aim of this study was to examine whether small biopsy specimens are representative of the whole human skeletal muscle or whether the different fibre types are unevenly distributed at different depths of the muscle. Ten micrometre thick cross-sections of whole human tibialis anterior were prepared using LKB PMV Cryo-Microtomes with a stroke length of 160 to 480 mm and the sections were stained for myofibrillar ATPase according to a modified procedure. The total and relative number of different fibres (Types 1 and 2) was determined in every 9th mm2 of the section. The data obtained were analysed by means of a computer program, which allowed assessment of bivariate data in the form of contour plots. The total number of fibres varied greatly between individuals (from 96 000 to 162 000; five individuals). The relative number of different fibres varied systematically in all individuals as a function of depth in the muscle. There was a gradual, often dramatic, relative increase in Type 2 fibre occurrence from the surface of the muscle (about 10--25%) towards the deeper regions (30--50%), the maximum being approximately along a line slightly posterior to the middle of the muscle. Additionally, superficial peaks were seen in places. In conclusion, the fibre type distribution in the tibialis anterior is not random. These results point to the importance of defining biopsy depth.

Evaluation of tissue preparation methods and paired immunofluorescence staining for immunocytochemistry of lymphomas

Abstract: Eight cross-linking fixatives were tested for preservation of extracellular or intracellular IgG, IgA, IgM, IgD, κ and ν light chains, J chain and secretory component. Most of the selected fixatives have been used in recent immunohistochemical studies of lymphoproliferative processes and comprised routine formalin, glutaraldehyde(1%)-formalin, Baker's formalin-calcium, formalin-sublimate, acetic acid(2%)-formalin-saline, Bouin's fluid, Susa fixative, and carbodiimide. The results obtained in artificial test substrates with defined amounts of IgG or IgA and in biological substrates (colon mucosa, tonsils, and different types of lymphomas) were compared by immunofluorescence with the antigenic preservation afforded by fixation in cold 96% ethanol (with or without inclusion of a pre-fixation 48 h washing period). An antigen concentration at least an eight-fold higher was necessary for detection with most other fixatives. Bouin's and Susa fixatives were peculiar in that they required antigen concentration 150 times higher for detection of IgG but only 3–8 times higher for IgA. Light chains were relatively well preserved by all fixatives except glutaraldehyde. For all cross-linking fixatives, the extent of antigenic masking depended on the concentration of environmental proteins, and the efficiency of unmasking with pronase or trypsin, therefore, varied with the location in the tissue. The J chain was particularly vulnerable to degradation during proteolytic treatment. The extensive masking of extracellular immunoglobulin in formalin-fixed tissue afforded a relatively good signal-to-noise ratio for immunoglobulin-producing cells when κ and ν chains were traced. Thus, differentiation between polyclonal and monoclonal B-cell processes on the basis of cytoplasmic labelling was often better in undigested sections. However, the light-chain type of membrane immunoglobulin could usually not be determined in directly fixed tissue. Ethanol fixation preceded by washing in saline afforded such determination and also preserved certain T-cell and HLA-DR antigens as well as diffuse α-naphthylbutyrate esterase. Reactive and malignant macrophages could further be traced by their cytoplasmic expression of L1 antigen, both in formalin- and ethanol-fixed material.

The role of plant hormones in higher plant cellular differentiation. II. Experiments with the vascular cambium, and sclereid and tracheid differentiation in the pine,Pinus contorta

Abstract: In sterile-cultured explants of stems of the pinePinus contorta Dougl., fusiform cambial cells differentiated entirely into axial parenchyma cells when exogenous indol-3yl-acetic acid (IAA) was omitted. The normal appearance of the cambial zone was maintained when IAA was included in the medium. The IAA-maintained stability of cambial structure suggests physiological rather than epigenetic control over vascular cambium structure.

Effects of histological processing on lectin binding patterns in oral mucosa and skin.

Abstract: The effects of fixation and wax processing on lectin binding to C3H mouse palate and tail skin were evaluated using eight FITC-conjugated lectins. Sections and blocks of tissue were fixed in acetone, ethanol, methanol, formalin, glutaraldehyde or Bouin's picric-acetic-formalin fixative. Tissue blocks were then processed to paraffin wax.

On the nature of Romanowsky--Giemsa staining and its significance for cytochemistry and histochemistry: an overall view

Abstract: The chances of Romanowsky-Giemsa (RG) staining becorning a reliable and useful histochemical procedure are reviewed, based on the now proven fact that RG staining requires two dyes only, namely, cationic Azure B and anionic Eosin Y. These two dyes differe from otherwise similar dye combinations in that they give, on distinct biological substrates, one additional colour, purple, which cannot be obtained by the use of either dye alone. The purple colour characterizes the Romanowsky-Giemsa effect (RGE), which is the essential feature of RG staining.

An assessment of the value of epithelial membrane antigen and other epithelial markers in solving diagnostic problems in tumour histopathology

Abstract: Using standard immunohistological techniques on formalin-fixed paraffin-embedded sections, we have evaluated the role of epithelial membrane antigen in the histopathological diagnosis of tumours. Of the 70 samples examined, 22 were taken for staging purposes from patients known to have carcinoma and, in half these cases, malignant cells were seen which could not be identified with confidence by conventional means. Forty-eight tumours were stained in order to determine their histogenesis. Twenty-two of these were positive and 20 subsequently proved to be epithelial on follow-up studies, including six in which a diagnosis of non-epithelial malignancy had been made on conventional preparations. The distinction of anaplastic carcinoma from malignant lymphoma and of spindle-cell carcinoma from sarcoma were the most useful applications. One of the positive tumours was of germ cell origin and in one the histogenesis is still not clear. Comparison with carcinoembryonic antigen and pre-keratin showed that epithelial membrane antigen was the most sensitive marker of epithelial differentiation in formalin-fixed tissue. A combination of all three reagents, though, increases diagnostic accuracy and allows tentative suggestions to be made about the possible site of origin of a metastatic carcinoma.

Repeated application of first-layer antiserum improves immunofluorescence staining: a modification of the indirect immunofluorescence staining procedure.

Abstract: Repeated application of the first-layer antiserum in the indirect immunofluorescence technique considerably improves the immunostaining. The modified method reveals more antigenic sites and increases the contrast between specific and background stainings, particularly where sparsely distributed antigenic areas are to be investigated. The effect of this novel immunostaining procedure is compared with that of the routine procedure and of other modifications. Possible mechanisms for the improving effect are discussed. A procedure of combined modifications is recommended for exploration of non-abundant antigens or for achieving a high-quality photograph.

Combined immunohistochemical staining for surface IgD and T-lymphocyte subsets with monoclonal antibodies in human tonsils.

Abstract: The aim of the present paper is to detect two different antigens simultaneously in a single slide. In cryostat sections of human tonsils, B-lymphocytes of follicle mantle-bearing surface IgD were immunostained with the alkaline phosphatase method using monoclonal anti IgD. The subsequent staining for T-lymphocyte subsets (T-helper and T-suppressor lymphocytes) was performed again with the alkaline phosphatase method using one of the monoclonal antibodies OKT 4, OKT 8, Leu 3a, Leu 2a. The best results with the alkaline phosphatase method were achieved using naphthol AS phosphate and Fast Blue BB for the revelation of the first antigen and naphthol AS-BI phosphate and diazotized New Fuchsin for the second.

Cytophotometry of glucose-6-phosphate dehydrogenase activity in individual cells.

Abstract: With the aid of thin films of polyacrylamide gel containing purified glucose-6-phosphate dehydrogenase subjected to cytochemical procedures for the enzyme using tetranitro blue tetrazolium, arbitrary units of integrated absorbance obtained with a Barr & Stroud GN5 cytophotometer were converted into units of enzyme activity. This conversion enabled cytochemical data to be compared directly with biochemical values. The conversion was applied to the cytochemical estimation of glucose-6-phosphate dehydrogenase activity in isolated rat hepatocytes, mouse oocytes, rabbit thymocytes, human granulocytes and human fibroblasts. Several control procedures were performed to confirm the admissibility of this conversion, such as: the estimation of the absorption characteristics of the formazans of tetranitro blue tetrazolium both in solution and precipitated in biological specimens; the linearity of the relationship between the increase of absorbance and incubation time; and the effect of different incubation conditions on the amount of specific formazan production.

A microscope-based flow cytophotometer.

Abstract: By means of a new flow chamber, a standard fluorescence microscope with Epi illumination and 100 W mercury arc excitation has been turned into a flow cytophotometer combining high resolution and sensitivity with simplicity of operation. In the flow chamber, cells are passed in a narrow stream through the microscope focus carried by a laminar flow of water running on the open surface of a cover glass which is coupled to the oil immersion microscope objective. Two spectral components of the fluorescence, for example, resulting from specific staining of two different cellular constituents with different dyes, can be measured simultaneously in separate channels so as to produce three-dimensional histograms. The scattered light of the cells is detected in dark field by a second microscope situated opposite the primary objective. Scattered light detection is integrating with regard to scattering angle from 0° to 90°. Hence, diffraction pattern effects are eliminated and the light scatter signal is approximately proportional to cell dry weight. The Epi illumination, which implies that excitation and fluorescence collection are parfocal, greatly simplifies instrument adjustment, which is further facilitated by the fact that the cell stream can be viewed at high magnification. Cell measuring time is about 3 μs which implies a measuring rate of 3×103 cells/s at 1% coincidence rate. Sensitivity is sufficient for measuring the DNA content of bacteria (that is, approximately 5×10−15 g/cell) with a coefficient of variance (CV) of about 6%. CV<1% is achieved for DNA histograms of mammalian cells. A 5 W argon laser as excitation source facilitates slit scan analysis and increases the sensitivity and measuring rate by one to two orders of magnitude.

Immunohistochemical differentiation between histiocytic and lymphoid neoplasms.

Abstract: Lysozyme has, until recently, been accepted as the only reliable immunohistochemical marker of benign and malignant histiocytes. Using this marker, very few lymphoreticular neoplasms of histiocytic origin are recognized and more recently α-1-anti-trypsin has been shown to be a better marker of malignant histiocytes. By immunizing rabbits with highly purified human blood monocytes we have obtained an antiserum (S22) which stains histiocytes and neutrophils in paraffin sections with a high degree of specificity. Using this antiserum and antisera to lysozyme and α-1-anti-trypsin we have stained paraffin sections of tissues containing reactive histiocytes, histiocytic proliferations, leukaemic infiltrates and lymphoreticular tumours of histiocytic and T-cell origin. Our results show that α-1-anti-trypsin is the most reliable marker of malignant histiocytes but that the as yet uncharacterized antigen defined by S22 may offer a promising alternative.

Ultrastructural localization of actin in muscle, epithelial and secretory cells by applying the protein A-gold immunocytochemical technique

Abstract: Actin-immunoreactive sites have been localized at the electron microscope level by the protein A-gold technique in striated and smooth muscle cells as well as in epithelial and secretory cells. The combination of the highly sensitive protein A-gold technique with the good ultrastructural preservation and retention of antigenicity obtained using low-temperature embedding conditions has allowed a very precise identification of the labelled structures with high resolution. In striated muscle cells the labelling was obtained over the myofilaments and the Z-band, mainly at its periphery. Labelling was also observed at the edge of the intercalated discs of the cardiac muscle cells. In smooth muscle cells the labelling was present over the myofilaments; the dense plaques associated with the plasma membrane were labelled at their periphery where actin filaments have been reported to anchor. In epithelial cells of the duodenum and the renal convoluted proximal tubule, the labelling occurred over the filamentous core of the microvilli and over the cell web. Gold particles were often present over, or closely associated with, the cell membrane at the tip of the microvilli or of invaginations and vesicular structures. At the level of the junctional complexes the gold particles were aligned at the edge of the dense zones. In pancreatic endocrine and exocrine secretory cells, actin-immunoreactive sites were revealed over the Golgi apparatus, mainly at the level of the inner cisternae in the maturing face over or closely associated with the membranes of the condensing vacuoles and secretory granules, and also over the plasma membrane. Microvilli and cell web were also labelled. Finally, in fibroblasts, gold particles were associated with the membrane of vesicular structures. The consistent finding of actin-immunoreactive sites closely associated with membranes of secretory granules and vesicular structures brings support to the proposal that contractile proteins might play an important role in transcellular transport and protein secretion.

Investigation of relationships between collagens, elastin and proteoglycans in bovine thoracic aorta by immunofluorescence techniques.

Abstract: Types I, III and V collagens and proteoglycan were localized in the aorta by indirect immunofluorescence techniques. Type I collagen was more prominent in media and adventitia than in intima while type III collagen predominated in intima and media but appeared less significant in adventitia. Type V collagen was observed in intima and media only and was seen surrounding smooth muscle cells. Type I collagen was located between elastic fibres but type III collagen appeared to envelop the fibres, suggesting an interaction between elastic fibres and type III collagen. Pretreatment of sections with testicular hyaluronidase caused no changes in staining for type I collagen, but adventitial areas showed increased staining for type III collagen. After digestion with chondroitinase ABC, intimal and medial areas showed increased staining for type III collagen. Therefore, type III collagen forms stronger interactions with proteoglycans and hyaluronic acid than does type I collagen and type III collagen in adventitia is largely masked by hyaluronic acid, while type III collagen in intima and media is associated with proteoglycan. Thus, type III collagen is a more significant component of adventitia than previously recognized. Proteoglycan was also partly localized along elastic fibres. It is, therefore, suggested that elastic fibres are coated with type III collagen, which itself is coated with proteoglycan.

Some recent developments in immunocytochemistry

Abstract: Immunocytochemistry has become an indispensable tool both in basic biomedical research and in diagnostic histophathology. Several recent innovations have led to improvements in the sensitivity, specificity and precision of these techniques.

DNA, RNA, protein and heterochromatin changes during embryo development and germination of soybean (Glycine max L.).

Abstract: DNA, RNA, protein and heterochromatin were measured cytophotometrically in developing soybean (Glycine max) seeds. The average 2C DNA content for the soybean genome was 2.64 pg. The amounts of nuclear DNA in embryo axes showed no significant change during embryo development, whereas the DNA content in cotyledon nuclei increased significantly from 3.58 pg to 5.49 pg. The number of endopolyploid nuclei increased from 26% to 48% and the DNA content from 4.45 to 5.49 pg after cessation of cell division. The changes in RNA and protein content during embryo development were in general similar to those in DNA content. This can be interpreted that increased DNA levels in soybean cotyledons generated during embryogeny increase the protein synthesizing capacity. During the first 15 days of germination, the number of endopolyploid nuclei in cotyledons declined from 46% to 4%, and this decline is interpreted as DNA degradation providing a ready source of nucleosides and phosphates during early embryo growth. A later decline, however, between 15 and 20 days after germination, was age related similar to leaf senescence, because the percentage of endopolyploid nuclei remained unchanged while the number of non-viable cells increased. In senescing cotyledons, 73% and 80% of RNA and protein but only 20% of DNA were lost, as compared to dormant cotyledons. The heterochromatin (condensed chromatin) measurements indicated that nuclei of metabolically inactive dormant and senescent cotyledon nuclei contained an average of 33% more heterochromatin than nuclei from the green cotyledons of seedlings.

Cytochemical changes in the nucleus during tumour development.

Abstract: The general background to tumour analytic work using quantitative optical cytochemical methods is first presented. An instrument complex, constructed especially for multiparameter work in cytopathological material has been developed. Nuclear changes have been followed in cell populations during their development through different grades of atypia to cancer and conspicuous cytochemical changes were observed. In a comprehensive series of clinically verified mammary carcinomas, a large percentage of cases was found in which the DNA values were within the normal range, while the others showed pronounced aneuploidy. A clear correlation was found between DNA profile-type and patient survival, the latter of which reflects the degree of malignancy in the individual case. The shift from resting state (G0) to growth activated G1-stage is initiated by a large increase of the nuclear proteins. Mammary tumours of a high malignancy grade, as judged by their DNA profile type, showed an especially great accumulation of nuclear protein and thus a high degree of activation. DNA-profile measurements, preferably combined with determinations of nuclear proteins can thus be used for judging malignancy grades in mammary tumours, which is also of considerable clinical interest. An as yet limited observational material also indicates similar situations in some other types of tumour.

Cytochemical properties of euchromatin and heterochromatin.

Abstract: Two classic cytochemical tests, the Feulgen-Schiff reaction and Toluidine Blue basophilia, have been employed for investigating the differential characteristics of heterochromatin and euchromatin. Differences have been detected in the Feulgen hydrolysis kinetics, the Feulgen absorption spectrum, the image analysis of Feulgen-stained material, and the binding of Toluidine Blue under ordinary and Mg2+ competitive staining conditions. The differences are assumed to be a function of the composition and stereo-arrangement of the DNA and DNA-protein complexes present in these chromatin types and are possibly associated with physiological activities whose whole meaning is far from being clear. Differences in optical retardations in Toluidine Blue-stained material were also found. These are interpreted as being due to chromatin packing state and selective removal of histones promoted by the acetic acid-ethanol fixative.

Localization and identity of adenosine deaminase-positive cells in tissues of the young rat and calf

Abstract: Rabbit antibody to calf adenosine deaminase (ADA) was used to localize this enzyme in tissues of the young rat and calf by the immunoperoxidase method. The distribution patterns of ADA in most tissues were similar for both species. Within the thymus gland, the enzyme was strongly expressed predominantly in cortical lymphocytes. In the spleen and lymph nodes, most lymphocytes of T-cell areas stained weakly for ADA, whereas only a small number of ADA-positive cells were found in B-cell areas. Clumps of strongly ADA-positive mononuclear blastoid and plasma cells were observed in the medullary regions of lymph nodes, around peri-arteriolar lymphocyte sheaths and in the red pulp of the spleen, and in the lamina propria of the intestine. Double immunofluorescence staining studies in the rat showed that some of these blastoid cells contained both ADA and immunoglobulins and appeared to be plasmablasts. Strong staining for ADA was also found, in both the rat and calf, in as yet unidentified mononuclear blastoid cells in the interstitium of non-lymphoid organs (kidney, heart, lung), in endothelial cells of some arterioles and capillaries, and in Kupffer cells of the liver. In addition, ADA was strongly expressed in calf bile canaliculi. These studies define areas in rat and calf tissues which contain ADA-positive cells and provide a model system for investigations of the relationship between ADA and the function and development of these cells.

Cytochemical evidence for functional zonation of parietal cells within the gastric glands of the mouse

Abstract: Parietal cells in the luminal segments of mouse gastric glands show high activity of acid-secreting potassium-dependent adenosine triphosphatase (H+, K+-ATPase) and of nicotinamide adenine dinucleotide-linked isocitrate dehydrogenase (NAD-ICDHase) and malate dehydrogenase (MDHase) but low activity of succinate dehydrogenase (SDHase). This pattern of activity is reversed in the basal segments of the same glands. These results and previous morphological findings support the conclusion that luminal segment parietal cells are much more active in hydrochloric acid secretion than those of the basal segment. The origin of this zonation may be either cellular deterioration with age or some more specific form of regulation of parietal cell metabolism.

Histochemistry of complex carbohydrates in the hairy skin of the domestic pig

Abstract: The hairy skin from eleven body areas of the domestic pig has been studied by means of a series of selected methods for the detection of complex carbohydrates, including peroxidase-labelled lectin-diaminobenzidine procedures. The results obtained were most distinctive in the areas where the skin accessories were involved. The complex carbohydrates in the epidermis appear to be acidic and neutral glycoproteins containing a small amount of sialic acid residues. The dermal connective tissue fibres revealed positive reactions for neutral and acidic glycoproteins with notable amounts of sialic acid residues, and for proteoglycans. In different parts of the hair follicle, neutral and acidic glycoproteins were also present, most being restricted to the connective tissue sheath and the dermal papilla. In the lower regions of the outer epithelial root sheath the epithelium contained a large amount of glycogen. The sebaceous glands reacted very weakly for neutral and acidic glycoproteins. The apocrine glands exhibited mostly neutral but also acidic glycoproteins in the secretory cells and the lumenal secretion, with substantial amounts of various saccharide residues.

Cuprolinic Blue: a specific dye for single-stranded RNA in the presence of magnesium chloride. II. Practical applications for light microscopy.

Abstract: The application of the new nucleic acid dye Cuprolinic Blue to cell smears and tissue sections has been described. Without added cations, Cuprolinic Blue stains both DNA and RNA, whereas in the presence of 1m MgCl2, Cuprolinic Blue specifically staing single-stranded RNA only. The total RNA can be stained after removal of DNA by DNAase digestion. Fixation in a modified Carnoy solution gave optimal staining results in all cases tested. By cytophotometry, a reliable and reproducible relative estimate can be obtained of the total nucleic acid content, the total RNA content and the amount of single-stranded RNA alone per cell.

Cuprolinic Blue: a specific dye for single-stranded RNA in the presence of magnesium chloride. I. Fundamental aspects

Abstract: Qualitative and quantitative aspects of the cationic dye Cuprolinic Blue were investigated with model films of polyacrylamide gel in which RNA, DNA and other biological polyanionic compounds had been incorporated. In the presence of 1m MgCl2, Curpolinic Blue was found to bind specifically to single-stranded RNA, leaving native DNA, proteins, (acid) polysaccharides and phospholipids completely unstained. Under these conditions, Cuprolinic Blue is complexed by non-electrostatic bonds with non-stacked purine bases, mainly adenine. Optimal conditions for dye binding and differentiation have been defined. Both the Cuprolinic Blue-MgCl2 staining of single-stranded RNA and the Cuprolinic Blue staining of RNA and DNA in the absence of MgCl2 were found to obey the Lambert-Beer law. The advantages and possible applications of Cuprolinic Blue are compared with well-known (indirect) histochemical RNA staining procedures.

The role of plant hormones in higher plant cellular differentiation. I. A critique

Abstract: Primary growth and morphogenesis in higher plants can be explained mechanistically in terms of primary types of cellular differentiation, namely, phenomena of cell division, primary wall growth, intercellular bonding and polarity. Plant hormones fulfil essential roles in regulating these types of differentiation, and it is well established that plant hormones can initiate primary growth and morphogenesis. Secondary and terminal types of cellular differentiation largely determine the usefulness of plants to man; however, regulators of these types remain poorly characterized. Secondary and terminal types need not differentiate in order for primary growth and morphogenesis to occur, and there is no conclusive evidence that factors regulating primary growth and morphogenesis also initiate subsequent types of differentiation.

The intracellular localization of amiloride in frog skin

Abstract: The diuretic compound amiloride is often used as a specific inhibitor of the passive Na+ entry step in the transepithelial transport of Na+ across frog skin. We have utilized the fluorescence properties of amiloride to study the distribution of this transport inhibitor in the ventral skin ofRana pipiens. After a 30 s exposure of 1–100 μm amiloride to the external surface of frog skin, amiloride fluorescence was evident in the cytoplasm of all cell layers of the epidermis and alveolar gland epithelium. Changes in the conditions of incubation which alter the pharmacological activity of amiloride did not affect the intracellular distribution of amiloride or the washout profile of [14C]amiloride. The presence of amiloride fluorescence in the cytoplasm prevented our examination of changes in the amiloride fluorescence at the cell surface with various conditions of incubation. Four derivatives of amiloride that differed in their ability to inhibit short-circuit current were also localized intracellularly but varied in their relative distribution among the cell layers of the epidermis. Our results indicate that when incubated at concentrations from 1 to 100 μm, a large fraction of the amiloride taken up by frog skin is not directly involved with the inhibition of passive Na+ transport at the apical surface of the stratum granulosum. The mechanism of intracellular uptake of amiloride is not clear. However, the cytoplasmic localization of amiloride could explain the action of the drug on intracellular enzymes and may account for the large proportion of non-displaceable [14C]amiloride that has been observed in frog skin.

Studies on the phenazine methosulphate-tetrazolium salt capture reaction in NAD(P)+-dependent dehydrogenase cytochemistry. I. Localization artefacts caused by the escape of reduced co-enzyme during cytochemical reactions for NAD(P)+-dependent dehydrogenases.

Abstract: The correct localization of oxidative enzymes using cytochemical tetrazolium methods, in which low molecular weight electron carriers such as NAD(P)H and reduced phenazine methosulphate (PMSH) are used, can be endangered by the escape of the reduced intermediates before they react to form the insoluble formazan at the true enzyme-containing sites. To investigate this phenomenon, the glucose-6-phosphate dehydrogenase reaction was studied in fixed erythrocytes which, because of their microscopic dimensions, are well-suited for studying the loss of intermediates. A mixture of active and heat-inactivated fixed erythrocytes was incubated in a PMS-supplemented medium for glucose-6-phosphate dehydrogenase. The cytophotometric histograms showed that the final formazan precipitate was equally distributed over both active and inactivated cells. When bovine serum albumin was added to the medium, all the formazan was found to be bound to this protein and the erythrocytes remained essentially unstained. The false localization in this system could be explained by an unfavourable balance between the capture of electrons carried by NADPH within the erythrocyte and the diffusion of NADPH out of the erythrocyte. The rate constant of NADPH oxidation was determined, as was also the diffusion constant of NADPH in a protein matrix. Substituting the data obtained into formulae derived from the enzyme cytochemical localization theory of Holt & O'Sullivan (1958), it was calculated that the capture reaction was highly deficient and, theoretically, less than 1% of the total amount of formazan produced was localized within the erythrocyte which explains the false localization observed. The importance of these findings for the cytochemical demonstration of NAD(P)+-dependent dehydrogenases in cells and electropherograms is briefly discussed.

Studies on the phenazine methosulphate-tetrazolium capture reaction in NAD(P)+-dependent dehydrogenase cytochemistry. II. A novel hypothesis for the mode of action of PMS and a study of the properties of reduced PMS.

Abstract: The results in the preceding paper have shown that the PMS-tetrazolium capture reaction as such is not sufficient to guarantee a correct localization of formazan in microscopically small dehydrogenase sites. For cytochemical reactions where the application of PMS leads to increased formazan formation, it is proposed that PMS functions not on its own, but as an efficient acceptor of NAD(P)H-oxidizing flavoproteins and thus increases the local NAD(P)H tetrazolium oxidoreductase activity. For the redox mediator vitamin K3 this type of mechanism could be proven with rat liver fractions. The relatively rapid NADPH oxidation precluded such simple experiments with PMS. An indication of such a stimulation by PMS was, however, obtained with soluble rat liver fraction. As escape of reducing equivalents from the site might also occur at the level of reduced PMS (PMSH) the solubility properties of PMSH were studied. It was found that PMSH has a low solubility in aqueous media and is hydrophobic. On basis of these findings a 'post-tetrazolium reduction' method seemed possible and could be experimentally confirmed.