Showing papers in "Histochemical Journal in 1986"

Improvement in the staining and in the visualization of the argyrophilic proteins of the nucleolar organizer region at the optical level.

Abstract: The argyrophilic proteins of the nucleolar organizer region (Ag-NOR proteins) were specifically localized at the optical level with a modified one-step silver technique performed at 20° C.

Aminoalkylsilane-treated glass slides as support for in situ hybridization of keratin cDNAs to frozen tissue sections under varying fixation and pretreatment conditions.

Abstract: Attempts to investigate the cellular localization of keratin mRNAs byin situ hybridization with specific [35S]-labelled cDNA probes to mouse epithelia have been seriously impeded by the uncontrollable detachment of frozen tissue sections on conventionally coated glass slides (i.e. those coated with egg white, gelatine, collagen). Similarly, a variety of other coating and attachment devices have proved to be unsatisfactory or impracticable for large scale investigations. These difficulties were completely overcome andin situ hybridization was possible after a short immersion of the glass slides in a 2% solution of 3-aminopropyltriethoxysilane in acetone. This treatment provides the glass surface with aminoalkyl groups which are apparently able to react covalently with aldehyde or ketone functions of frozen tissue sections. The resulting firm adhesion of the sections enabled us to investigate the influence of different fixation and prehybridization procedures on the quality of thein situ hybridization. It was found that especially harsh prehybridization, involving hydrochloric acid, heat and proteinase K treatment, drastically reduces the morphological integrity of the sections, thus rendering a reliable assignment of the label difficult. In contrast, a mild prehybridization, consisting mainly of a rehydration of the sections in phosphate-buffered saline and equilibration in 0.1 M glycine, leaves the morphology intact and leads to a highly efficient and specificin situ hybridization.

In situ hybridization methods for the detection of somatostatin mRNA in tissue sections using antisense RNA probes.

Abstract: In situ hybridization studies with [32P] and [3H] labelled antisense RNA probes were undertaken to determine optimal methods of tissue fixation, tissue sectioning, and conditions of hybridization, and to compare the relative merits of the two different radioactive labels. The distribution of somatostatin mRNA in neurons of rat brain using a labelled antisense somatostatin RNA probe was employed as a model for these studies. The highest degree of sensitivity forin situ hybridization was obtained using paraformaldehyde fixation and vibratome sectioning. Optimal autoradiographic localization of mRNA was obtained within 7 days using [32P] labelled probes. However, due to the high energy emittance of [32P], precise intracellular localization of hybridization sites was not possible. [3H] labelled RNA probes gave more precise cellular localization but required an average of 18–20 days autoradiographic exposure. The addition of the scintillator, PPO, decreased the exposure time for the localization of [3H] labelled probes to seven days. We also report a method for combinedin situ hybridization and immunocytochemistry for the simultaneous localization of somatostatin in mRNA and peptide in individual neurons.

Differential expression of keratin 19 in normal human epithelial tissues revealed by monospecific monoclonal antibodies.

Abstract: Three monospecific monoclonal antibodies (BA16, BA17 and A53—B/A2) recognizing different epitopes of the human keratin 19 were used to determine tissue distribution of this 40 kDa keratin polypeptide. Immunohistochemical methods revealed four different staining patterns among normal human epithelial tissues: firstly, complete negativity of the epidermis, sebaceous glands, hepatocytes and other tissues; secondly, homogeneous positivity as seen for example in the gall bladder and urinary bladder epithelium, endometrium and many other epithelia; thirdly, a mosaic of positive and negative cells among mammary gland luminal cells, prostate epithelia and some other epithelia and fourthly, a more complex heterogeneous pattern found in non-keratinizing squamous epithelia and hair follicles with generally the basal layer being the most strongly or sometimes exclusively stained. The pattern seen in non-keratinizing squamous epithelia varied considerably according to the fixation method and the antibody used as well as among different donors and in different areas of the same organ. The other three staining patterns were on the other hand nearly identical with all three antibodies on both frozen sections and sections of methacarn-fixed paraffinembedded tissues. Our results provide evidence for differential expression of the human keratin 19 at the single cell level, an observation which could be exploited in the study of epithelial differentiation and pathology.

Presence of chromogranin A, B and C in bovine endocrine and nervous tissues: A comparative immunohistochemical study

Abstract: Antisera against chromogranin A, B and C were used to study the distribution of these acidic proteins in bovine endocrine and nervous tissues. The three chromogranins occur together in several endocrine organs (adrenal medulla, anterior pituitary, endocrine pancreas) and in sympathetic ganglion cells. In the posterior pituitary, only chromogranin C and in the intermediate lobe only A and C are found. The parathyroid gland contains only A, and enterochromaffin cells are immunoreactive for A and B. Cells of the thyroid gland and some cells of the anterior pituitary apparently do not contain any chromogranins. It is concluded that the three chromogranins are not always stored together and that they are not present in all endocrine cells. This distinct localization of the chromogranins indicates some special, although still undiscovered, function for these proteins.

Aminomethyl coumarin acetic acid: a new fluorescent labelling agent for proteins.

Abstract: A new fluorescent protein labelling agent, 7-amino-4-methyl coumarin-3-acetic acid (AMCA), emits in the blue region (440-460 nm) on activation with UV light (350 nm). The active reagent is the N-hydroxysuccinimide ester which reacts with lysine residues under mild conditions to form photostable amide links. The Stokes shift of 100 nm compared to 30 nm for Fluorescein isothiocyanate (FITC) allows easy filter discrimination of exciting and emitting radiation. The agent has been demonstrated in use for fluorohistochemical examination of human kidney glomeruli, using the sandwich technique and compared with the same procedure using FITC-labelled antibodies. The good quantum yield coupled with convenient emission lines in the mercury spectrum allows photographic exposure time of fluorescent labelled sections to be reduced to a quarter of that required for a corresponding FITC conjugate. AMCA-immunoglobulin conjugates were not susceptible to photobleaching and have a storage life at -20 degrees C of more than two years.

The effects of mechanical stability on the macromolecules of the connective tissue matrices produced during fracture healing. I: The collagens

Abstract: The distribution of types I, II, III, V and IX collagens in healing fractures of the rabbit tibia has been demonstrated by immunofluorescent techniques. It has also been shown that the mechanical stability of the healing fracture affects both the distribution and types of the collagens present. The initial fibrous matrix contains types III and V collagens; type I collagen was only located in this matrix if unfixed tissue was used. In mechanically stable fractures, cancellous bone forms over the entire periosteal surface by 5–7 days; type I collagen is laid down within the previous fibrous matrix. The trabeculae are heterogeneous in their collagen content. The cavities contain a matrix of types III and V collagens. Small nodules of cartilage may be present between 7 and 14 days; these contain types II and IX collagens. In mechanically unstable fractures, cancellous bone is initially formed away from the fracture gap. The fibrous tissue over the gap is replaced by cartilage; types II and IX collagens are laid down on the pre-existing fibrous matrix. The cartilage is replaced by endochondral ossification. At the ossification front, type I collagen is found around the chondrocyte lacunae of the spicules of cartilage. The new trabeculae contain a core of cartilage which is surrounded by a bone matrix of types I and V collagens. The fracture gaps are invaded by fibrous tissue, which contain types III and V collagens. This is later replaced by cancellous bone.

Differentiation of histochemically visualized mercury and silver

Abstract: Accumulations of silver and mercury can be visualized in tissue sections by a technique called autometallography or physical development. In order to make a histological differentiation between mercury and silver in tissue exposed to both metals, it is necessary to remove one of the metals while leaving the other untouched. The present paper describes a technique by which silver accumulations in histological sections can be removed by potassium cyanide, yet leaving mercury accumulations intact to be developed autometallographically.

Immunogold localization of ingested kidney bean (Phaseolus vulgaris) lectins in epithelial cells of the rat small intestine

Abstract: The interactions between dietary kidney bean (Phaseolus vulgaris) lectins and the epithelial cells of the rat small intestine were investigated by immunogold electron microscopy. The results demonstrated that the lectins bind to the glycocalyx of duodenal and jejunal microvilli and that some of these dietary constituents are endocytosed into lysosomal pathways within both absorptive and secretory gut cells. It is concluded that the lysosomal response serves to limit the absorption of nutritionally significant levels of these dietary toxins.

The compartmentation of glycolytic and gluconeogenic enzymes in rat kidney and liver and its significance to renal and hepatic metabolism.

Abstract: An indirect immunoperoxidase procedure has been used to demonstrate sites of glycolysis and gluconeogenesis in normal rat kidney and liver. In kidney, the gluconeogenic enzyme fructose 1,6-biphosphatase was restricted to the proximal tubular epithelium, while the glycolytic enzyme hexokinase predominated in more distal segments. Intense staining for the biphosphatase in proximal convoluted tubular brush borders suggests that reabsorbed substrates may be used directly at this site in renal gluconeogenesis. In view of the high phosphofructokinase and pyruvate kinase activities present in collecting ducts, their relatively low hexokinase activities and their relatively pale immunostaining for hexokinase indicate that glycolytic substrates which feed into the pathway subsequent to the initial phosphorylation step, rather than glucose, may be the major energy source for the rat renal papilla.

The out-of-range error in microdensitometry.

Abstract: Reaction rates of enzyme activity in tissue sections or individual cells can be determined directly by the continuous monitoring of the final reaction product as it is formed us ing cytophotometric equipment. Studies of glucose-6-phosphate dehydrogenase activity (Butcher, 1984; Butcher & Van Noorden, 1985) have shown that the rates of such reactions are often nonlinear due to at least, two phenomena. Firstly, the test reaction is usually too high because it includes the so-called 'nothing dehydrogenase' reaction; this is particularly evident during the first few minutes of the reaction (Butcher & Van Noorden, 1985; Van Noorden et aI., 1985). Subtraction of the proper control from the test reaction permits the determination of the true reaction rate for the enzyme. In the case of glucose-6-phosphate dehydrogenase, the proper control reaction is that occurring in the absence of substrate and coenzyme (Butcher & Van Noorden, 1985), Secondly, nonlinearity can be caused by tailing off in the test reaction at higher absorbance values. It has been explained by either clogging of the active sites of the enzyme by the final reaction product, thus slowing down the reaction (Pette, 1981), or a heterogenous distribution of the reaction product within the scanning spot of the cytophotometer, the 'out-of-range' error (Goldstein, 1981). According to Butcher (1984) and Butcher & Van Noorden (1985), a linear 'test-minus-control' reaction rate is possible until the intensity of the test reaction reaches a certain mean integrated absorbance value. At higher absorbance values, the reaction tails off due to an inbuilt cut-off in the linear response of the microdensitometer (Goldstein, 1981). When reaction rate studies are performed with a Vickers M85 scanning and integrating microdensitometer, the cutoff can be expected when local absorbance values of 1.1 have been reached in the spot to be measured, and therefore tailing off starts to occur at a mean integrated absorbance value of approximately 0.7 (Butcher, 1984). Recently, a new Vickers M85a microdensitometer was installed in the laboratory of one of us (CJFVN). One of the advantages of this newer version is a linear response up to an absorbance value of approximately 2.0 (Fig. 1). Therefore, the hypothesis that it is the out-of-range error which causes nonlinearity at higher absorbance values has been tested with this new machine. Linearity should still occur at mean integrated absorbance values much higher than 0.7 Cryostat sections, 12 #m thick, of mature female rat liver were incubated on the stage of the Vickers M85a microdensitometer at 26 ° C in a reaction medium containing 100 mM phosphate buffer, pH 7.45, 32% w/v polyvinyl alcohol Grade G04/140, 10 mM glucose-6-phosphate,

A post-embedding avidin-biotin peroxidase system to demonstrate the light and electron microscopic localization of lectin binding sites in rat kidney tubules

Abstract: Summary A post-embedding method for the light and electron microscopic demonstration of lectin binding sites in rat kidney tubules is described. The use of biotinylated lectins, followed by treatment with avidin peroxidase and the DAB-H 202 sequence, produced intense staining of acrylic sections at the electron microscope level: brush borders and associated structures, cytoplasmic granules, basal infoldings and basement membrane-plasmalemmal interfaces of proximal tubules bound erythrophytohaemagglutinin, while distal tubules were mainly unstained. At the light microscope level, epoxy resin sections showed a similar staining pattern after etching, as did acrylic resin sections after intensification.of the final reaction product. The binding of wheatgerm agglutinin to cytoplasmic granules and brush border structures in the proximal tubules was abolished, at both the light and electron microscope levels, by the competing sugar tri-N-acetylchitotriose. Epoxy resin ultrathin sections required etching before staining was achieved in the electron microscope, and results were far inferior to those obtained with acrylic resin. This method allows rapid and inexpensive screening of large numbers of tectins, if required, at both the light and electron microscope levels, using reagents that are stable for long periods of time.

The distribution of sympathetic nerve fibres in the AV node and AV bundle of the bovine heart

Abstract: The sympathetic nervous system has important effects on the properties of the heart, including the conduction of the impulse. However, it is not known how this nervous system is distributed in the atrioventricular (AV) bundle, which together with the AV node constitutes the only conduction pathway between the atria and ventricles in normal hearts. Therefore, in the present study the adrenergic innervation in the bovine AV node/AV bundle was examined by use of the glyoxylic acid induced method for histofluorescence demonstration of catecholamines. Acetylcholinesterase (AChE) histochemistry was also used. It was found that the AChE-positive nerve fascicles in these regions partly contain sympathetic nerve fibres, that sympathetic nerve fibres occur in the proximity of some of the ganglionic cells that occur outside the AV node/AV bundle, that the arteries supplying AV bundle tissue as well as AV nodal tissue have perivascular plexuses of sympathetic nerve fibres, and that there is a substantial number of sympathetic nerve fibres outside Purkinje fibre bundle surfaces. The observations give new insight into the question of the distribution of the sympathetic nerves in the AV bundle in relation to the distribution of these nerves in the AV node. Possible functional implications of the observations are discussed.

Merkel cell (small cell) carcinoma of the skin: immunohistochemical and ultrastructural demonstration of distinctive perinuclear cytokeratin aggregates and a possible association with B cell neoplasms.

Abstract: Three cases of Merkel cell (small cell) carcinoma of the skin are presented with immunohistochemistry for epithelial and neuroendocrine antigens. All three cases showed distinctive punctate perinuclear cytoplasmic positivity for cytokeratin which corresponded to aggregates of intermediate filaments, seen ultrastructurally in two cases. Epithelial membrane antigen was also identified in two cases. Only one case showed cytoplasmic positivity for neuron specific enolase, and immunostaining for a battery of polypeptide hormones was negative. The demonstration of cytokeratin perinuclear inclusions provides a distinctive immunohistochemical feature to aid in their diagnosis. Two of the three patients had chronic lymphocytic leukaemia years before the diagnosis of Merkel cell carcinoma. The possible association of lymphoproliferative disorders, particularly B cell tumours, and Merkel cell carcinoma is discussed.

A quantitative histochemical study of NADPH-ferrihemoprotein reductase activity.

Abstract: A quantitative histochemical assay for NADPH-ferrihemoprotein (P450) reductase had been developed For optimal activity, it is necessary to use a relatively electropositive tetrazolium salt such as neotetrazolium chloride as the final acceptor The apparentKm of the reaction is 083 mM Its specificity has been proven in two ways: (i) activity is increased selectively in the pericentral zone of liver from rats treated with phenobarbitone, an inducer of the reductase, though not in liver of rats injected with 3-methylcholanthrene, which induces NAD(P)H dehydrogenase; (ii) it is competitively inhibited by NADP+ (Ki=150mm) though unaffected by dicumarol, an inhibitor of NAD(P)H dehydrogenase activity An NADP+ concentration ten times greater than the substrate concentration inhibits the histochemical reaction and the reaction in a microsomal fraction assayed biochemically to the same degree (70% inhibition) The amount of inhibition is independent of temperature, of the zone of the acinus and of the treatment of the animal

Use of pectinases complexed to colloidal gold for the ultrastructural localization of polygalacturonic acids in the cell walls of the fungus Ascocalyx abietina.

Abstract: Three pectinase—gold complexes were used to localize polygalacturonic acids in the fungusAscocalyx abietina (Lagerberg) Schlaepfer-Bernhard. With the pectinesterase and pectin lyase—gold complexes, the labelling was uniformly distributed over the fungus walls and did not seem to be significantly influenced by the tissue preparation. With the polygalacturonase—gold complex, differences in the labelling distribution were noted according to the fixation procedure indicating, therefore, that osmication of the tissues could greatly interfere with the localization of the specific enzyme binding sites. These results demonstrate, for the first time, the possibility of detecting polygalacturonic acids by means of different gold-complexed pectinases.

Immunohistochemical localization of cell surface receptors using a novel method permitting simple, rapid and reliable LM/EM correlation.

Abstract: A method is presented which allows correlative serial section analysis by light and electron microscopy of cell surface antigens in monolayer cultures. Sites of antigenicity are shown by deposition of diaminobenzidine after pre-embedding, immunoperoxidase immunocytochemistry. Osmication is replaced by the use of gold chloride which specifically enhances the electron density of diaminobenzidine. In addition gold chloride bound to diaminobenzidine survives embedding and provides the basis for a post-embedding photochemical amplification method. Immunostained cells are embedded in LR White by a rapid technique which preserves their structure and leaves them available for subsequent post-embedding immunocytochemistry. The method is illustrated by the demonstration of epidermal growth factor (EGF) receptors on the EGF receptor-rich human carcinoma cell line A431 using a well characterized monoclonal antibody raised against EGF receptor.

Calcium-stimulated myofibrillar ATPase activity correlates with shortening velocity of muscle fibres in Xenopus laevis

Abstract: The iliofibularis muscle ofXenopus laevis is reported to contain five types of fibres which have different force—velocity relationships. Ten fibres of each type were selected on the basis of succinate dehydrogenase activity, cross-sectional area and location in the muscle, in order to assess the validity of the fibre type classification.

Arylsulphatase activity associated with phenanthrene induced digestive cell deletion in the marine mussel Mytilus edulis.

Abstract: The acid hydrolase arylsulphatase has been localized at the ultrastructural level in digestive cells of the marine musselMytilus edulis for control and phenanthrene-treated (200µg/l) animals. In untreated mussels the activity was generally restricted to the lysosomal—vacuolar system and the Golgi apparatus. It was associated with all types of vesicle, although not all individual vesicles were reactive. In heterolysosomes which were filled with precipitate the reaction product was most densely associated with the limiting membranes. Lipid inclusions commonly occurred in the digestive cells; these sometimes showed limited reaction for enzyme activity. The striking difference between normal and phenanthrene-treated samples was the presence in all treated animals of reaction product in the inter-cellular spaces and varying degrees of cytoplasmic activity in a number of digestive cells. This is interpreted as a sign of impending cell deletion. Sections for morphological examination showed evidence of increased digestive cell deletion in phenanthrene-treated mussels. The process results in release of membrane-bound bodies into the tubule lumen.

Histochemical detection of glycogen usingGriffonia simplicifolia agglutinin II

Abstract: The utility of a lectin fromGriffonia simplicifolia (GSA II) for demonstrating glycogenin situ was tested on fixed paraffin-embedded sections of a variety of tissues from rodents and man. The histochemical specificity of GSA II conjugated to horseradish peroxidase (GSA II—HRP) for glycogen was documented by the lability of such staining on adjacent sections treated with either malt diastase orα-amylase. In some tissue sites, the lectin—HRP conjugate imparted cytoplasmic staining that was diastase- and amylase-labile but resisted digestion withN-acetylglucosaminidase. Reactivity in the latter sites was attributed to glycogen and occurred in cell types having well-documented glycogen content, including liver hepatocytes, skeletal muscle fibres and polymorphonuclear leukocytes. In other tissue loci, GSA II binding was confined to cell surfaces or intracellular compartments, was not affected by prior diastase or amylase digestion, but was abolished withN-acetylglucosaminidase. Staining in these sites was attributed not to glycogen, but instead to glycoconjugate having sugar chains terminated withN-acetylglucosamine. These findings document the affinity of GSA II for glycogenin situ but do not conflict with the biochemically demonstrated affinity of GSA II for terminalN-acetylglucosamine. The results reported here show that the GSA II—HRP method may be useful for detecting physiological or pathological changes in the glycogen content of cells.

Zonal analysis of metabolic profiles of articular-epiphyseal cartilage chondrocytes: a histochemical study.

Abstract: Articular—epiphyseal cartilage from the femur of New Zealand rabbits was subjected to histochemistry for determination of the presence of metabolic enzymes along its zonal stratification Glycolytic enzymes were strongly reactive in all of the zones Krebs cycle enzymes, enzymes of the hexose monophosphate shunt and the respiratory chain enzymes showed a progressive increase in reactivity from the tangential zone through the top half of the epiphyseal zone Indicators of lipid metabolism were fairly high in all regions of the cartilage

Recent advances in the detection and characterization of specific antibody-forming cells in tissue sections.

Abstract: A new general approach has been developed for the detection of one or more different specific antibody producing cells and the simultaneous determination of their Ig isotype in tissue sections, after immunization of animals. Specificity of intracellular antibodies is demonstrated after incubation of the sections with an antigen-enzyme conjugate and the isotype of the antibodies is determined using an anti-immunoglobulin (Fc chain-specific)-enzyme conjugate followed by histochemical revelation of the two different enzymes. The principles of the method, the required antigen- and antibody-enzyme conjugates and their application in single, double or triple staining studies are reviewed. The method allows the detection of specific antibody-forming cells against protein antigens as well as against haptens. By means of haptens such as trinitrophenyl (TNP), immune responses against thymus dependent, thymus independent, and particulate antigens can be studied. In a limited number of cases the method can also be used to study the localization of antigen-antibody complexes.

Glycoconjugates in the secretory epithelium of the chicken mandibular gland.

Abstract: In the secretory epithelium of the chicken mandibular gland, glycoconjugates have been studied by means of histochemical methods of light and electron microscopy. In light microscopy, a series of histochemical procedures have been employed which included lectin—peroxidase—diaminobenzidine methods and a digestion technique with neuraminidase orα-amylase. In electron microscopy, a battery of methods were used that corresponded to those employed in light microscopy. In the secretory cells of the chicken mandibular gland, vicinal diol- and sulphate-containing glycoconjugates with sialic acid,α-d-mannose,α-d-glucose andβ-d-galactose residues were visualized and the possible histophysiological significances of such glycoconjugates were discussed with special reference to the functions of the salivary gland.

Influence of embedding media in prolactin labelling with immunogold techniques.

Abstract: Immunogold labelling of prolactin in three different embedding media was compared. The polymeric prolactin in secretory granules was labelled in the three media, however, acrylic monomers (Lowicryl K4M and LR White) provided a more intense labelling with higher dilutions of the primary antibody, compared to the labelling in the epoxy resin (araldite). An intense labelling of monomeric prolactin in Golgi complex was detected only in acrylic embedments, and the labelling on the rough endoplasmic reticulum was significant only in LR White embedded tissues.

Chromosomal localization of genes by scanning electron microscopy using in situ hybridization with biotinylated probes: Y chromosome repetitive sequences.

Abstract: The feasibility of using scanning electron microscopy (SEM) to identify the position of specific DNA sequences was examined using a Y chromosome ‘specific’ probe (pHY2.1). Tests were carried out on chromosome spreads hybridizedin situ with biotinylated pHY2.1. Chromosomal sites of hybridization of the probe were localized by an indirect immunohistochemical procedure which resulted in a gold product which could be amplified by silver precipitation. In the SEM, the specific location of the probe was easily identified due to the enhanced signal produced by the gold—silver complex. The probe was localized both on the long arm of the Y chromosome and within interphase nuclei. It was found that SEM was more sensitive than light microscopy since the probe could be identified without silver amplification. With refinements to the technique, SEM could provide a useful method for high resolution localizing of unique DNA sequences (i.e. single copy genes).

Heterogeneous expression of two oncodevelopmental antigens, CEA and SSEA-1, in colorectal cancer

Abstract: Colorectal carcinomas are composed of heterogeneous cell subpopulations which may be instrumental in conferring metastatic potential and therapeutic refractoriness to these tumours. To assess cellular heterogeneity, the expression has been examined of two oncodevelopmental antigens, carcinoembryonic antigen (CEA) and stage-specific embryonic antigen 1 (SSEA-1), by double immunofluorescence microscopy on 11 human colorectal carcinomas. Although both antigens were expressed in each tumour, their regional and cellular locations differed considerably. SSEA-1 expression was rarely expressed in poorly differentiated cancers but was enhanced with increasing degrees of differentiation. CEA expression was independent of histological differentiation. SSEA-1 was expressed with similar frequency in cell membranes, cytoplasm, and glandular contents regardless of degree of differentiation. Cytoplasmic staining with CEA however, was limited to more poorly differentiated tumours. In normal mucosa remote from the tumours and transitional mucosa adjacent to them, SSEA-1 stained only a few lower crypts whereas CEA stained a majority of both upper and lower crypts. Although biochemical studies have indicated that the SSEA-1 epitope may reside on CEA molecules, the fact that colon cancer tissues express these two antigens quite heterogeneously suggests differences in antigenic processing which may be dependent upon the degree of cellular differentiation.

Immunohistochemical detection of glycogen phosphorylase isoenzymes in rat and human tissues

Abstract: Glycogen phosphorylase has at least three isoenzymes, i.e. muscle-, liver-, and brain-types. Antibodies have been raised against highly purified isoenzymes from rat muscle, liver and brain and found to react specifically to extracts from human muscle, liver and brain, respectively. Using these antibodies and the unlabelled antibody—enzyme method, each of the three isoenzymes has been localized in both rat and human tissues.

Polyamine cytochemistry: Comparisons between cytochemical, autoradiographic, immunocytochemical and chemical results in the prostate

Abstract: Results obtained with two newly developed fluorescence cytochemical methods for detecting the polyamines spermidine and spermine have been compared to autoradiographic localization of biosynthetically labelled polyamines, to immunocytochemical results obtained with antibodies directed against spermidine and spermine, and to chemical polyamine determinations using the rat prostate as a model tissue. Complete agreement between all five methods was obtained. Application of perchloric acid to formaldehyde-fixed sections of rat prostate strongly reduced theo-phthalaldehyde inducible and formaldehyde-fluorescamine inducible fluorescence characteristic of spermidine and spermine. Perchloric acid extracted 40% of tissue-bound polyamines from formaldehyde-fixed tissue sections, and molecules with the physicochemical characteristics of polyamines constituted 80–90% of all fluorescamine reactive molecules extracted. Our results therefore confirm the specificity of theo-phthalaldehyde and formaldehyde-fluorescamine methods for polyamine cytochemistry. As polyamines are strongly implicated in cellular growth regulation and cancer, simple and inexpensive techniques for polyamine histochemistry may be useful for interpreting the biological and pathophysiological roles of these molecules.

Phenotypic differentiation patterns of the human monocyte/macrophage system.

Abstract: In the present study phenotypic properties of non-stimulated and stimulated blood monocytes and of their normal macrophage derivatives were studied applying enzyme cytochemistry, isoenzyme analysis of acid esterase (EC 3.1.1.6), and immunohistochemical staining using a panel of newly established monoclonal antibodies specific for the monocyte/macrophage lineage. Certain marker profiles could be established for the various normal subpopulations within the monocyte/macrophage system, which were also observable in epithelioid cells and U-937 cell line considered as reactive and neoplastic differentiation variants of monocytes, respectively. Alveolar macrophages, in contrast to the other analysed monocyte/macrophage populations, showed a highly activated phenotype comparable to lymphokine stimulated blood monocytes and epithelioid cells. The results underline the concept that the adaptation of monocytes/macrophages to their particular microenvironment is of decisive importance for their definitive differentiation.