Cytochemical Reaction Product Research Articles

Experimental substantiation of the possibility of using the cytochemical parameters of peripheral blood to assess the functional state of the organism of the pilot

The activity of succinate (mitochondrial), lactate, α-glycerophosphate, glucose-6-phosphate peripheral blood lymphocytes in rats under the conditions of modeling acute and chronic gravitational stress was studied by quantitative spectrophotometry of cytochemical reaction products to reveal the localization of oxidative enzymes. The dynamics of morphofunctional transformations is determined, the severity of which depends on the duration of the effect (the rotation time of the animals on the centrifuge). For acute gravitational stress, changes in the activity of mitochondrial succinate dehydrogenase are characteristic, and in the various stages of chronic gravitational stress, glycolysis (increase in lactate dehydrogenase and α-glycerophosphate dehydrogenase activity) and plastic metabolism (increase in glucose-6-fosfata dehydrogenase activity) are characteristic. These changes are explained by the inclusion of neurohumoral regulatory mechanisms that ensure synchronization of oxidative metabolic processes in the cells of organs of various body systems under unfavorable conditions of their functioning. Significant correlation links between the activity of lymphocyte enzymes with the same indices of neurons of the celiac sympathetic node, adenocytes of the cortical and medulla of the adrenal glands have been established. This suggests that the activity of lymphocyte enzymes can be used as indirect indicators of the processes occurring in the organs of the sipato-adrenomedular and pituitary-adrenocortical systems under conditions of acute and chronic gravitational stress.

Cytoarchitectural features of Ucides cordatus (Crustacea Decapoda) hepatopancreas: structure and elemental composition of electron-dense granules

Hepatopancreal tissue of the crab Ucides cordatus was investigated by light and electron microscopy. The observed epithelial cells were: E-cells (embryonic), located in the distal portion of the hepatopancreal tubules, R-cells (resorptive) F-cells (fibrillar) and B-cells (blister or secretory), found in its intermediate and proximal regions. Two types of electron-dense granules (EDGs) were found frequently in the cells of the proximal portion of the hepatopancreal tubule. Both types of EDGs presented alternating concentric electron-dense and electron-lucent layers. In order to better characterize these granules, energy dispersive X-ray analysis (EDXA) and glucose-6-phosphatase (G6Pase) cytochemistry were performed. One type of spherical granule was seen inside vacuoles surrounded by an association of myelin-like membranes as well as some small membrane-bound vesicles. This type of granule neither presented detectable Ca and P on EDXA spectra nor G6Pase cytochemical reaction products. The second type of granule had O, P and Ca characteristic peaks. G6Pase cytochemical products were observed inside these structures and showed that this mineralized type was surrounded by endoplasmic reticulum membranes. This result suggests that in U. cordatus the endoplasmic reticulum is associated with the genesis of mineralized EDGs. While amorphous mineral granules may be associated with a storage of Ca and P for the new carapace synthesis, EDGs covered by the non-mineralized spherical multi-layered membranes may be associated with late endosomes. No specific secretory pathway however was determined for the EDGs at the epithelial proximal portion.

Sodium- and potassium-activated ATPase in the mammalian vestibular system

Autoradiographic and cytochemical procedures were employed to determine the cellular distribution of the Na,K-ATPase enzyme in the mammalian vestibular system. A light-microscope survey of vestibular tissues incubated with [ 3H]ouabain shows high densities of ouabain binding sites within the dark cell epithelium (DC) of the ampullae of the semi-circular canals, and to a lesser extent, the DC of the utricular macula. A moderate number of binding sites was found in nerve fibers penetrating the connective tissue beneath the sensory epithelium (SE) of the ampullae and the maculae. A small number of binding sites is distributed in the deep portion of the SE, both in the ampullae and in the maculae. These latter binding sites seem to be associated with nerve terminals and receptor cells. At the ultrastructural level, the vestibular dark cells exhibit extensive basolateral membrane infolding, a morphological hallmark of cells engaged in trans-epithelial ion transport. The cytochemical reaction product is K +-dependent, ouabain inhibitable, and is restricted to the basolateral membrane extensions, with little or no product on the luminal membrane. The extent of membrane infolding in dark cells of the utricle is less pronounced than that of the ampullar dark cells and the intensity of the cytochemical reaction appears to correlate with the extent of membrane infolding. The results support the widely held hypothesis that the vestibular dark cells play a role in endolymph production. They also suggest that the vestibular sensory epithelia may be a site of ion exchange.

Second Messengers in the Locomotor-Sensory System of First Multicellulars. The IP 3 -Containing Compartments in Chemoreceptor Cells of the Comb Jelly Beroe cucumis from Data of Cytochemical Study

Result of the performed electron microscopy cytochemical study was an indirect detection of intracellular localization of inositol 1,4,5-triphosphate according to sites of its binding with specific phosphatase in receptor and secretory cells of epithelium surrounding actinostome of the comb jelly Beroe cucumis. The highest level of the deposit of the enzymatic cytochemical reaction product was revealed in chemoreceptor cells of I type. In all cells of the epithelium as well as in nerve terminals and neuroplexus elements, the following compartments had the highest deposit content: cellular membrane and glycocalyx areas, microtubules, microfilaments, mitochondria, neurotubules, and structures of the synaptic apparatus. The obtained data expand the picture of cytochemical distribution and of the contents of the deposit compatible with localization of components of inositol pathway in receptor cells of the locomotor-sensory system of Beroe cucumis [1, 2] and indicate its essential role in the mechanism of chemosensory transduction.

Bile canalicular contraction and dilatation in primary culture of rat hepatocytes--possible involvement of two different types of plasma membrane Ca(2+)-Mg(2+)-ATPase and Ca(2+)-pump-ATPase.

Increasing evidence has indicated that bile canalicular contraction is mediated by the nonmuscular Ca(2+)-calmodulin-actomyosin system, and the contraction facilitates canalicular bile flow. The aim of the present study was to examine, by electron cytochemistry, how the expression of two types of plasma membrane Ca(2+)-ATPase, i.e., Ca(2+)-Mg(2+)-ATPase and Ca(2+)-pump-ATPase, is related to the dynamic changes of bile canalicular contraction. Hepatocytes isolated from male Wistar rat liver by collagenase perfusion were cultured to form a primary monolayer. The canalicular dynamics in the couplets and triplets were analyzed by time-lapse cinematography. The Ca(2+)-Mg(2+)-ATPase activity was identified by the electron cytochemical method of Ando. Ultrastructural localization of Ca(2+)-pump-ATPase was examined by immunogold electron microscopy. We found that cytochemical reaction products showing the presence of Ca(2+)-Mg(2+)-ATPase activity were localized on the luminal side of the bile canalicular membranes. Immunogold particles, indicating the presence of Ca(2+)-pump-ATPase, were located mainly on the cytoplasmic side of the bile canalicular membranes. The expression of both Ca(2+)-ATPases on the canalicular membranes was enhanced during the contracting stage of bile canaliculi, whereas their expression was diminished in the dilating stage. We conclude that two different types of bile canalicular Ca(2+)-ATPase may be involved in the regulation of canalicular contractility to control the extrusion of intracytoplasmic free calcium ions into the canalicular lumen.

Secondary Messengers in the Locomotor-Sensory System of Early Multicellulars. Inositol-Containing Compartments in Receptor Cells of Ctenophores and Coelenterates according to the Data of Cytochemical Investigation

The electron microscopy cytochemical data have been obtained on compartmentalization of a secondary messenger, inositol (1,4,5)-triphosphate (InsP3), in receptor cells of different modalities in representatives of early multicellular animals, ctenophores Beroё cucumis and coelenterates, scyphomedusas Cyanea arctica. In the gravitation mechanoreceptor cells of the ctenophores and medusas, the inositol-containing compartments were revealed predominantly in cilia and stereocilia. The presence of uniformly distributed discrete granules of the precipitate of the enzymatic cytochemical reaction product in the light-sensitive lamellar structures of photoreceptor cells of ctenophores is discussed in the connection with the possible existence of peculiar rhodopsin–inositol complexes participating in photoreceptor processes. The peculiarities of compartmentalization of the precipitate in photo- and gravitation mechanoreceptor cells allow considering InsP3 as one of the components that that are able to contribute to development of mechanisms of modality in the ciliary receptor cells of ancient multicellular animals.

Expression of plasma membrane Ca2+-ATPase on hepatic sinusoidal endothelial fenestrae: modification of the one-step method.

The intracytoplasmic free calcium ion (Ca2+) concentration is maintained at a low level in mammalian tissues by extruding Ca2+ against a high extracellular Ca2+ concentration, mainly through the activity of the plasma membrane Ca2+-ATPase pump. The objective of the present study was to localize the plasma membrane Ca2+-ATPase activity on hepatic sinusoidal endothelial cells (SECs) by electron microscopic cytochemistry. The ultrastructural localization of Ca2+-ATPase activity on ultrathin sections of liver tissue and cultured SEC monolayer was examined by the electron microscopic cytochemical method of Ando (method A: original method) and by our modified method (method B: shortened fixation method). By method A, scanty cytochemical reaction products of Ca2+-ATPase were found in the SECs. By method B, Ca2+-ATPase activity was clearly localized on the outer surface of the plasma membrane of sinusoidal endothelial fenestrae (SEF). Our modification of Ando's method by shortening the incubation time of liver tissue or isolated SEC sections in the substrate allowed clear demonstration of Ca2+-ATPase activity on the SEF membrane. Use of tangential sections of primary cultures of SEC provided excellent localization results. The cytochemically reactive Ca2+-ATPase expressed on the SEF plasma membrane may be involved in regulation of the intracytoplasmic Ca2+ concentration.

Cytochemical and ultrastructural studies of the scales of the Amoeba Cochliopodium bilimbosum (Testacea)

Summary Cochliopodium bilimbosum, a testacean amoeba, has characteristic scales on its dorsal surface. The structural components of these scales and the process of scale formation were examined by electron microscopy and also by cytochemical staining for polysaccharides. Abundant vesicles and vacuoles containing rudimentary scale-like structures were observed in the cytoplasm. During the transport of vacuoles from Golgi area to the dorsal surface of the organism, the scales became fully developed. Fine grains of the cytochemical reaction products were detected in the scales, and the intensity of staining increased during the transport of the vacuoles. These results suggest that the scale components are assembled in vesicles that are derived from the Golgi complex. The vesicles then fuse to form vacuoles and, during transport of the vacuoles to the dorsal surface, the organization of the scales may be completed both morphologically and biochemically.

The organization of the endoplasmic reticulum and the intermediate compartment in cultured rat hippocampal neurons.

The boundaries of the organelles of the biosynthetic endomembrane system are still controversial. In this paper we take advantage of the unique architectural organization of neurons to investigate the localization of a spectrum of compartment-specific markers with the goal of defining the location of the rough endoplasmic reticulum (ER), smooth ER, intermediate compartment, and the Golgi complex. Markers of the rough ER (signal sequence receptor), Golgi complex (mannosidase II), and the trans Golgi network (TGN38) were essentially restricted to the cell body and the initial segment of one of the cell's dendrites. In contrast the cytochemical reaction product for glucose 6 phosphate, a classical ER marker, in addition to staining ER structures in the cell body also reacted with smooth ER elements that extended into both axons and dendrites. These peripheral smooth ER elements also reacted at the immunofluorescence level for ER marker 3-hydroxy-3-methylglutaryl-coenzyme A reductase, as well as for calnexin and protein disulfide isomerase. We also analyzed the location of rab1, rab2, p58, the KDEL receptor, and beta-subunit of coatomer. These intermediate compartment markers were found predominantly in the cell body but also extended to the proximal parts of the dendrites. Collectively, our data argue that the ER of hippocampal neurons consists of functionally and spatially distinct and separated domains, and they stress the power of the hippocampal neuron system for investigations of the organization of the ER by light microscopy.

Multicolour preparations for in situ hybridization using precipitating enzyme cytochemistry in combination with reflection contrast microscopy

We have further developed a method for the detection of different enzyme cytochemical reaction products by means of reflection contrast microscopy (RCM). By embedding these enzyme precipitates in a protein matrix, we were able to prevent the reaction products from dissolving in immersion oil, which is required for RCM analysis. The applicability of the RCM procedure is, therefore, extended to a range of cytochemical enzyme precipitation methods, which normally result in oil soluble reaction products. To test their usefulness, these enzyme precipitates have been used in single- and well as double-label in situ hybridization (ISH) procedures to visualize a number of DNA target sequences by several different reflection colours, i.e. white, yellow and red. Three repetitive DNA probes for the (sub)centromeric regions of chromosomes 1, 7 and 17, as well as a repetitive DNA probe for the telomeric region of chromosome 1, and two cosmid DNA probes (40 kb each) for both arms of chromosome 11 could be detected with high efficiency in both interphase and metaphase preparations. Moreover the enzyme precipitates were shown to be stable upon exposure to excitation light or upon storage. It may be concluded that these findings render RCM a sensitive method for the visualization of multiple targets in biological specimens.

Blood platelet heterogeneity: evidence for two classes of platelets in man and rat.

The platelet population of man and rat can be divided into two classes of about equal size on the basis of presence/absence of an acid phosphatase which acts on para-nitrophenylphosphate (a PNPase), at pH 5. The cytochemical reaction product is in the platelet cytoplasmic matrix, without apparent association with organelles or membrane systems. We could not relate differences in staining to differences in function: all cells responded the same to activation by thrombin, ADP, or collagen, in fibrinogen binding to activated platelets, by endocytosis of fluid-phase tracers, and in internalization of latex particles. With respect to possible physiological substrates for the PNP-ase, there was no reaction product from beta-glycerophosphate, AMP, ADP, ATP, GTP, CMP, IMP, cAMP, creatine phosphate, and inositol phosphates, and the enzyme was not inhibited by 40 mM lithium. There was reaction product from tyrosine phosphate suggesting that the physiological substrate for PNP-ase is tyrosine phosphate. In rat bone marrow, megakaryocytes also were of two classes, PNPase positive and PNPase negative, suggesting that different classes of platelets arise from different classes of megakaryocytes.

Xeroxographic-assisted location of areas or structures on clinical slides for EM examination

Recent studies in our laboratories have resulted in the development of microwave-accelerated silver stains for the demonstration of basement membranes and fungi, gram-negative bacteria, type III collagen, endoneurium, perineurium, DNA, and DAB or PPD-PC cytochemical or immunocytochemical reaction products. We have also found that the very structure in a specimen that raised a question by light microscopy can be mapped with a Micro-Locator Slide and then located and examined by the SEI and BEI modes of SEM by the techniques collated in this summary.

Cytochemical detection of gene expression: Light and electron microscope evaluation of bacterial β-galactosidase reaction product

Recombinant DNA technology allows the introduction of genes into the genome of cells in vitro and in vivo. “Reporter” genes cotransfected with other genes of interest indicate successful transfection because cellular expression can be detected by histochemistry, autoradiography, or in situ hybridization histochemistry. The Escherichia coli gene, lacZ, is a frequently used reporter gene because it does not affect the usual functions or activities of the cell; the gene is present and expressed in successive generations; and bacterial β-galactosidase (β-gal), the enzyme produced as a result of expression of the gene, can be detected cytochemically. Studies that evaluate transfected cells among mixed cell populations in vivo, e.g. gene therapy and cell lineage studies, can benefit from the detectability of the cytochemical reaction product at the light and electron microscope levels. After incubation at pH 7.4 in X-gal (5-bromo-4-chloro-3 indolyl-β-D-galactoside), a commercially available indigogenic substrate, a blue-green reaction product results in cells that have synthesized functional bacterial β-galactosidase enzyme. This reaction product is visible in intact cells, and is retained during processing and embedment for thin section analysis in the transmission electron microscope. This report demonstrates the value of lacZ as a reporter gene using a clone of transfected HTD114 cells of human fibrosarcoma origin, selected for their high lacZ expression. Figure 1 is a dark field image of reaction product in 1 μm thick epon sections of cells that were incubated in X-gal, fixed, pelleted, osmicated, dehydrated, and embedded by routine methods. Figure 2 shows the appearance of the reaction product at the ultrastructural level. At both LM and EM levels, the reaction product varies in quantity among cells; appears focally concentrated in some cells, diffuse in others; and is confined to the cytosome with a noticeable ring-like distribution around the nucleus in yet other cells. Dark field analysis of sections is especially helpful for detecting relatively low quantities of reaction product. The distinctive ultrastructural appearance of the reaction product allows the unequivocal identification of cells expressing lacZ as well as the ultrastructural features of the cells. Ultrastructural characterization of the cell is often important for precise identification of cell type, especially in tissues such as lung in which there are many different cell types.

The application of electron spectroscopic imaging for quantification of the area fractions of calcium-containing precipitates in nervous tissue.

Energy-filtering transmission electron microscopy has been applied to the quantification of area fractions of calcium-containing cytochemical reaction products in central nervous tissue and the retina of fish. The method of electron spectroscopic imaging using electrons with an energy loss of 250 eV produces images with a very high, structure-sensitive contrast. This is a suitable imaging condition for the reliable detection of reaction products and structural details in unstained ultrathin sections. The images were recorded with a sensitive TV camera and evaluated with the integrated digital image-analysis system of the Zeiss CEM 902 energy-filtering electron microscope. An empirical procedure was developed which objectively detects reaction products and calculates characteristic values, taking into account different staining intensities. This new and sensitive method enabled an assessment to be made of the influence of temperature and light adaptation on cytochemically detectable calcium in nervous tissue of fish. Higher amounts of calcium-containing reaction product were detected in synaptic clefts of the optic tectum in warm-adapted fish than in cold-adapted fish. In synaptic vesicles of photoreceptor cells in the fish retina, higher amounts of reaction product were found in dark-adapted fish than in light-adapted fish.

Quantitative energy-filtered image analysis in cytochemistry: II. Morphometric analysis of element-distribution images

A combination of energy-filtered electron microscopy (EFEM) and an image-analysis system (IBAS/2000) is used for a morphometric analysis of chemical reaction products in cells. Electron energy-loss spectroscopic element-distribution images are acquired from cytochemical reaction products in a variety of cellular objects: (1) colloidal thorium particles in extra-cellular coat material, (2) iron-containing ferritin particles in liver parenchymal cells, (3) barium-containing reaction products in endoplasmic reticulum stacks, (4) elements present in lysosomal cerium- and barium-containing precipitates connected with acid phosphatase (AcPase) or aryl sulphatase (AS) enzyme activity. Areas or area fractions are determined from such element-distribution images by application of an objective image segmentation method. By superposition of two or more element-distribution images, mutual element relations are qualitatively established in lysosomal cerium- and barium-containing precipitates connected with acid phosphatase (AcPase) or aryl sulphatase (AS) enzyme activity. By comparing electron spectroscopic images (ESI) with element-distribution images, the mutual contrast per elemeny relations are quantitatively investigated. The obtained gain in resolution in such electron energy-loss spectroscopic element-distribution images will be explained and discussed.

The effect of tartrate on bone cell acid phosphatase activity: a quantitative cytochemical study.

Tartrate-resistant acid phosphatase activity (TRAPase) is widely used as a cytochemical marker to distinguish osteoclasts from macrophages and other related cell types. The degree of tartrate resistance, however, may depend on which reaction methods, tissues, or species are used. To investigate this further, we have measured the amount of cytochemical reaction product by microdensitometry. We compared osteoclast acid phosphatase (APase) activity in fresh frozen sections of neonatal rat calvaria using two different reaction methods; one is commonly employed for qualitative histochemistry and includes alpha naphthyl phosphate as substrate, simultaneous coupling to the chromagen Fast Garnet, and a 30-minute reaction time (method A). The other may be used to measure enzyme reaction rates in cells in situ and employs conditions suitable for initial velocity kinetics, namely naphthol-ASBI phosphate as substrate, post coupling to Fast Garnet, and a 2-minute reaction time. Although enzyme reaction rates differed greatly between the two methods, significant inhibition of APase activity by tartrate was observed in calvarial osteoclasts (69% and 59% with methods A and B, respectively), osteoblasts, and spleen macrophages. Using method B, mouse calvarial osteoclasts had similar APase activity to that seen in the rat. Tartrate produced little inhibition in these mouse cells, in contrast to the observations made with rat tissue, but murine spleen macrophages were significantly tartrate sensitive (40% inhibition with tartrate). On this basis, conclusions regarding the cell specificity of TRAPase should be treated cautiously.

Plateau absorbance measurements: an alternative approach to enzyme activity determination illustrated by the example of alkaline phosphatase.

A simple and reproducible method was used for the cytophotometric assay of alkaline phosphatase activity by end point measurements after incubation at 70 degrees C. Alkaline phosphatase was incorporated in polyacrylamide gel model films and its activity was demonstrated with a simultaneous coupling method. The initial reaction rate was 4.7 times faster than at 37 degrees C. At 37 degrees C, linear reaction rates were obtained up to 90 min incubation. Deviation from linearity occurred only when the amount of final reaction product precipitated inside the films was too high to be measured cytophotometrically. In that case, levelling off of the reaction rate was due to the out-of-range error of the cytophotometer. At 70 degrees C, reaction rates were distinctly non-linear from the onset of incubation. This was due to heat inactivation of the enzyme molecules. A plateau level was reached after approximately 60 min incubation, irrespective of the amount of enzyme incorporated, indicating that all enzyme molecules had become inactivated after this incubation period. The inactivation process followed first-order kinetics. The plateau value as well as the slope of the initial reaction were found to be linearly related to the amount of enzyme incorporated. Therefore, plateau absorbance values can be used as a relative measure of enzyme activity instead of initial reaction rates. This type of measurement could be valuable for routine applications of enzyme cytochemistry in diagnostic pathology, or when cytochemical reaction products are used as markers in immunocytochemistry or hybridocytochemistry. Precise control of incubation time is not necessary once the plateau value has been reached and preparations can be mounted and measured later.

Ultrastructural localisation of creatine kinase activity in the contact sites between inner and outer mitochondrial membranes of rat myocardium

The mitochondrial isoenzyme of creatine kinase, together with the ADP/ATP translocase, most probably belongs to a functional multi-enzyme complex located on the inner mitochondrial membrane. The outer membrane is a necessary constituent of this microcompartment. On the other hand, electron microscopic visualisation demonstrated the formation of contact sites between inner and outer mitochondrial membranes as a reaction to variations of the energy metabolism. In search for a possible correlation between these biochemical and morphological phenomena, rat myocardia were brought into the required energy state by stimulation through catecholaminergic mechanisms or adjusted perfusion with amytal. Subsequently, creatine kinase was cytochemically localised. Creatine kinase activity is demonstrated in membrane contacts between inner and outer mitochondrial membranes. The extent of contact sites and creatine kinase activity depends on the metabolic state as shown by morphometric analysis of the surface density of cytochemical reaction product. This surface density diminishes drastically after inhibiting the metabolic activity with amytal. It is concluded that these contact sites are dynamic micro-environments in which the active site of creatine kinase, oxidative phosphorylation and ADP/ATP transport interact during basal and stimulated metabolism.

Long clinostation influence on the localization of free and weakly bound calcium in cell walls of Funaria hygrometrica moss protonema cells

The pyroantimonate method was used to study the localization of free and weakly bound calcium in cells of moss protonema of Funaria hygrometrica Hedw . cultivated on a clinostat (2 rev/min). Electroncytochemical study of control cells cultivated at 1 g revealed that granular precipitate marked chloroplasts, mitochondria, Golgi apparatus, lipid drops, nucleoplasma, nucleolus, nucleus membranes, cell walls and endoplasmic reticulum. In mitochondria the precipitate was revealed in stroma, in chloroplast it was found on thylakoids and envelope membranes. The cultivation of protonema on clinostat led to the intensification in cytochemical reaction product deposit. A considerable intensification of the reaction was noted in endomembranes, vacuoles, periplasmic space and cell walls. At the same time analysis of pectinase localization was made using the electroncytochemical method. A high reaction intensity in walls in comparison to that in control was found out to be a distinctive pecularity of the cells cultivated on clinostat. It testifies to the fact that increasing of freee calcium concentrations under conditions of clinostation is connected with pectinic substances hydrolysis and breaking of methoxy groups of pectins. Data obtained are discussed in relation to problems of possible mechanisms of disturbance in calcium balance of plant cells and the role of cell walls in gomeostasis of cell grown under conditions of simulated weighlessness.

Pitfalls in ecto-5'-nucleotidase enzyme cytochemistry as demonstrated by the immunogold-labelling technique on macrophages.

We have used both the enzyme cytochemical method with lead nitrate as a capture agent and an immunological method at the electron microscope level to localize plasma membrane 5'-nucleotidase in rat peritoneal resident macrophages during the initial interactions of latex beads or heat-killed Escherichia coli with the cell during phagocytosis. In macrophages at rest, cytochemical reaction product was evenly distributed along the external surface of the plasma membrane. However, when the cells were phagocytosing latex beads or bacteria, reaction product covered the entire surface of the adhering particles. To determine whether the apparent redistribution of 5'-nucleotidase onto the adhering particle was fact or artifact, we localized 5'-nucleotidase using a monoclonal antibody and an immunogold labelling technique. In macrophages binding or beginning to ingest bacteria, gold particles were distributed along the plasma membrane, except at the sites of cell-bacterium internalization. More significantly, the adhering bacteria were free of gold particles and therefore had no 5'-nucleotidase on their surfaces. Latex beads proved to be unsuitable as a test particle because the gold particles stuck to them non-specifically. We conclude that the artifactual redistribution of lead-phosphate reaction product is a major drawback of enzyme cytochemical methods when used on cell surfaces and that the immunogold labelling technique is more reliable.