Summary
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.
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References
BENDAYAN, M. & SHORE, G. C. (1982) Immunocytochemical localisation of mitochondrial proteins in the rat hepatocyte.J. Histochem. Cytochem. 30, 139–47.
CARLEMALM, E., GARAVITO, R. M. & VILLIGER, W. (1982) Resin development for electron microscopy and an analysis of embedding at low temperature.J. Microsc. 126, 123–43.
CHANG, J. P. & YOKOYAMA, M. (1970) A modified section freeze-substitution technique.J. Histochem. Cytochem. 18, 683–4.
DE, MEY, J., MOEREMANS, M., GELLENS, G., NUYDENS, R. & DE, BRABANDER, M. (1981) High resolution light and electron microscopic localisation of tubulin with the IGS (immuno gold staining) method.Cell Biol. Int. Rep. 5, 889.
GILLETT, R. & GULL, K. (1972) Glutaraldehyde—its purity and stability,.Histochemie 30, 162–7.
HALMI, N. S. (1981) Immunoperoxidase staining of primate pituitaries with antibodies against the 3 subunits of human pituitary glyco-protein hormones.J. Histochem. Cytochem. 29, 837–43.
HARDY, P. H., PETRALI, J. P. & STERNBERGER, L. A. (1970) Post-embedding staining for electron microscopy by the unlabelled antibody enzyme method.J. Histochem. Cytochem. 18, 678.
HSU, S. M., RAINE, L. & FANGER, H. (1981) The use of avidin-biotin peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabelled antibody (PAP) procedure.J. Histochem. Cytochem. 29, 577–80.
ITO, S. & KARNOVSKY, M. J. (1968) Formaldehyde/glutaraldehyde fixatives containing trinitro compounds.J. Cell Biol. 39, 168a-9a.
JASANI, B., NEWMAN, G. R., THOMAS, N. D. & WILLIAMS, E. D. (1982) Hapten-labelled ligands as immunocytochemical probes of membrane and subcellular receptors.J. Path. 137, 80(P).
JASANI, B., THOMAS, N. D., NEWMAN, G. R. & WILLIAMS, E. D. (1983) DNP-hapten sandwich staining (DHSS) procedure: design, sensitivity, versatility, and applications.Immuno. Commun. (in press).
JASANI, B., WYNFORD-THOMAS, D. & WILLIAMS, E. D. (1981) Use of monoclonal antihapten antibodies for immunolocalisation of tissue antigens.J. clin. Path. 34, 1000–2.
LARSSON, L. (1979) Simultaneous ultrastructural demonstration of multiple peptides in endocrine cells by a novel immunocytochemical method.Nature, Lond. 282 743–6.
MAZURKIEWICZ, J. E. & NAKANE, P. K. (1972) Light and electron microscopic localisation of antigens in tissues embedded in polyethylene glycol with a peroxidase labelled antibody method.J. Histochem. Cytochem. 20, 969–74.
MORIARTY, G. C. & HALMI, N. S. (1972) Electron microscopic localisation of the adrenocorticotropin-producing cell with the use of unlabelled antibody and the peroxidase-antiperoxidase complex.J. Histochem. Cytochem. 20, 590–603.
MOZDZEN, J. J., JR & KEREN, D. F. (1982) Detection of immunoglobulin A by immunofluorescence in glycol methacrylate embedded human colon.J. Histochem. Cytochem. 30, 532–5.
PEASE, D. C. (1964) Morphological features of aldehyde fixation. InHistological Techniques for Electron Microscopy (edited by PEASE, D. C.), 2nd edn, p. 61. New York, London: Academic Press.
ROMANO, E. L., STOBINSKI, C. & HUGHES-JONES, N. C. (1974) An antiglobulin reagent labelled with colloidal gold for use in electron microscopy.Immunochemistry 11, 521–2.
ROTH, J., BENDAYAN, M., CALEMALM, E., VILLIGER, W. & GARAVITO, M. (1981) Enhancement of structural preservation and immunocytochemical staining in low temperature embedded pancreatic tissue.J. Histochem. Cytochem. 29, 663–71.
ROTH, J., BENDAYAN, M. & ORCI, L. (1978) Ultrastructural localisation of intracellular antigens by the use of protein A-Gold complex.J. Histochem. Cytochem. 26, 1074–81.
ROTH, J. & BERGER, E. G. (1982) Immunocytochemical localisation of galacto-syltransferase in HeLa cells: Codistribution with thiamine pyrophosphatase in trans-Golgi cisternae.J. Cell. Biol. 93, 223–9.
STERNBERGER, L. A. (1972) The unlabelled antibody-peroxidase and the, quantitative immunouranium methods in light and electron immunohistochemistry. InTechniques of Biophysical and Biochemical Morphology (edited by GLICK, D. and ROSENBAUM, R. M.), Vol. 1, p. 67. New York: Wiley.
STERNBERGER, L. A. (1979)Immunocytochemistry, 2nd edn. New York: Wiley.
TAKAMIYA, H., BODEMER, W. & VOGT, A. (1978) Masking of protein antigen by modification of amino groups with carbobenzoxychloride (benzyl chloroformate) and demasking by treatment with non-specific protease.J. Histochem. Cytochem. 26, 914–20.
WYNFORD-THOMAS, D., STRINGER, B. & NEWMAN, G. R. (1981) Hydroxyethylmethacrylate embedding: an improved technique.Med. Lab. Sci. 38, 121–2.
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Newman, G.R., Jasani, B. & Williams, E.D. A simple post-embedding system for the rapid demonstration of tissue antigens under the electron microscope. Histochem J 15, 543–555 (1983). https://doi.org/10.1007/BF01954145
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DOI: https://doi.org/10.1007/BF01954145