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-killedEscherichia 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.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
BLOK, J., ONDERWATER, J. J. M., DE WATER, R. & GINSEL, L. A. (1982) A cytochemical method for the demonstration of 5′-nucleotidase in mouse peritoneal macrophages with cerium ions used as trapping agent.Histochemistry 75, 437–43.
BOORSMA, D. M. (1984) Direct immunoenzyme double staining applicable for monoclonal antibodies.Histochemistry 80, 103–106.
BOZDECH, M. J. & BAINTON, D. F. (1981) Identification of α-naphthyl butyrate esterase as a plasma membrane ectoenzyme of monocytes and as a discrete intracellular membrane-bounded organelle in lymphocytes.J. Exp. Med. 153, 182–95.
CORDELL, J. L., FALINI, B., ERBER, W. N., GHOSH, A. K., ABDULAZIZ, Z., MACDONALD, S., PULFORD, K. A. F., STEIN, H. & MASON, D. Y. (1984) Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase (APAAP complexes).J. Histochem. Cytochem. 32, 219–29.
COURTOY, P. J., PICTON, D. H. & FARQUHAR, M. G. (1983) Resolution and limitations of the immunoperoxidase procedure in the localization of extracellular matrix antigens.J. Histochem. Cytochem. 31, 945–51.
CRAMER, E., WERB, Z. & BAINTON, D. F. (1984) Redistribution of ecto-5′nucleotidase during phagocytosis by guinea pig polymorphonuclear leukocytes.J. Leuko. Biol. 36, 463–75.
DE MEY, J. (1984) Colloidal gold as a marker and tracer in light and electron microscopy.E.M.S.A. Bull. 14, 54–60.
DE PIERRE, J. W. & KARNOVSKY, M. L. (1974) Ectoenzymes of the guinea pig polymorphonuclear leukocyte. I. Evidence for an ectoadenosine monophosphatase, adenosine triphosphatase andp-nitrophenyl phosphatase.J. Biol. Chem. 249, 7111–20.
EDELSON, P. J. (1981) Plasma membrane ectoenzyme: Macrophage differentiation markers. InHeterogeneity of Mononuclear Phagocytes, (edited by FORSTER, O. & LANDY, M.), pp. 143–9. New York: Academic Press.
EGUCHI, M., SANNES, P. L. & SPICER, S. S. (1979) Alterations in ultrastructural cytochemistry of complex carbohydrates of rat periotoneal macrophages during phagocytosis.J. Reticuloendo. Soc. 25, 207–18.
ESSNER, E. Phosphatases. (1973) InElectron Microscopy of Enzymes. Principles and Methods, Vol. 1 (edited by HAYATT, M. A.), pp. 44–76. New York: Van Nostrand Reinhold Co.
FAHIMI, H. D. (1980) InTrends in Enzyme Histochemistry and Cytochemistry. Ciba Found. Symposium Publ. 73, pp. 33–51. Excerpta Medica, Amsterdam.
GINSEL, L. A., DE WATER, R., VAN DER MEER J. W. M. & DAEMS, W. TH. (1985) Heterogeneity of 5′-nucleotidase activity and wheat-germ agglutinin binding in mononuclear phagocytes. InMononuclear Phagocytes: Characteristics, Physiology and Function. (edited by R. VAN FURTH), pp. 99–113. Boston: Martinus Nijhoff.
GOLDFISCHER, S., ESSNER, E. & NOVIKOFF, A. B. (1964) The localization of phosphatase activities at the level of ultrastructure.J. Histochem. Cytochem. 12, 72–95.
JANCKILA, A. J., LUNG, M. S., YAM, L. T. & LI, C.-Y. (1985) Immunoalkaline phosphatase cytochemistry: Technical considerations of endogenous phosphatase activity.Amer. J. Clin. Pathol. 84, 476–80.
JEMMERSON, R., KLIER, F. G. & FISHMAN, W. H. (1985) Clustered distribution of human placental alkaline phosphatase on the surface of both placental and cancer cells: Electron microscopic observations using gold-labeled antibodies.J. Histochem. Cytochem. 33, 1227–34.
LUZIO, J. P. & STANLEY, K. K. (1983) The isolation of endosome-derived vesicles from rat hepatocytes.Biochem. J. 216, 27–36.
MATSUURA, S., ETO, S., KATO, K. & TASHIRO, Y. (1984) Ferritin immunoelectron microscopic localization of 5′-nucleotidase on rat liver cell surface.J. Cell Biol. 99, 166–73.
NOVIKOFF, A. B. (1980) DAB cytochemistry: Artifact problems in its current uses.J. Histochem. Cytochem. 28, 1036–8.
PEARSE, A. G. E. (1980)Histochemistry: Theoretical and Applied, 4th edn.Comparative and Optical Technology. Vol. 1, pp. 159–62. New York: Churchill Livingstone.
POLAK, J. M. & varndell, i. m. (1984)Immunolabelling for electron microscopy Amsterdam: Elsevir.
RIEMER, B. L. & WIDNELL, C. C. (1975) The demonstration of a specific 5′-nucleotidase activity in rat tissues.Arch. Biochem. Biophys. 171, 343–7.
ROBINSON, J. M. & KARNOVSKY, M. Y. (1983) Ultrastructural localization of 5′-nucleotidase in guinea pig neutrophils based upon the use of cerium as capturing agent.J. Histochem. Cytochem. 31, 1190–6.
ROMANO, E. L. & ROMANO, M. (1984)Historical aspects in Immunolabelling for Electron Microscopy, (edited by POLAK, J. M. & VARNDELL, I. M.), pp. 3–15. Amsterdam: Elsevier.
SIDDLE, K., BAILYES, E. M. & LUZIO, J. P. (1981) A monoclonal antibody inhibiting rat liver 5′-nucleotidase.F.E.B.S. Lett. 128, 103–7.
STANLEY, K. K., BURKE, B., PITT, T., SIDDLE, K. & LUZIO, J. P. (1983) Localization of 5′-nucleotidase in a rat liver cell line using a monoclonal antibody and indirect immunofluorescent labelling.Exp. Cell. Res. 144, 39–46.
STENBERG, P. E., SHUMAN, M. A., LEVINE, S. P. & BAINTON, D. F. (1984) Redistribution of alpha-granules and their contents in thrombin-stimulated platelets.J. Cell Biol. 98, 748–60.
STEWART, D. I. H. & CRAWFORD, N. (1983) Redistribution of membrane 5′-nucleotidase in rabbit peritoneal polymorphonuclear leucocytes during phagocytosis.F.E.B.S. 156, 329–34.
STOWARD, P. J. (1980) InTrends in Enzyme Histochemistry and Cytochemistry. Ciba Found. Symposium Publ. 73, pp. 11–31. Excerpta Medica, Amsterdam.
WERB, Z. & COHN, Z. A. (1972) Plasma membrane synthesis in the macrophage following phagocytosis of polystyrene latex particles.J. Biol. Chem. 247, 2439–46.
WIDNELL, C. C. (1972) Cytochemical localization of 5′-nucleotidase in subcellular fractions isolated from rat liver. I. The origin of 5′-nucleotidase activity in microsomes.J. Cell Biol. 52, 542–58.
About this article
Cite this article
Cramer, E.M., Werb, Z. & Bainton, D.F. Pitfalls in ecto-5′-nucleotidase enzyme cytochemistry as demonstrated by the immunogold-labelling technique on macrophages. Histochem J 20, 108–116 (1988). https://doi.org/10.1007/BF01746611
- Gold Particle
- Test Particle
- Lead Nitrate
- Resident Macrophage
- Latex Bead