Abstract
Antibodies, in general, provide the most sensitive and specific methods for detecting the protein products of genes. Immunoperoxidase techniques described here detect 103-105 molecules/cell (depending on whether the protein is dispersed within the cell or concentrated at high density in a particular compartment); with various enhancement methods, this can be improved more than tenfold. Immunohistochemistry is particularly suitable for analyzing transfection in vitro, because it examines individual cells; methods based on analysis of bulk RNA (e.g., Sl nuclease protection [see Chapter 21], this volume]) or protein (e.g., immunoblotting) are considerably less sensitive when only a small proportion of cells are transfected. Immunoperoxidase reactions are generally more sensitive than immunofluorescence (Chapter 27) and immunogold methods and require no special optics to observe. Also, the reaction product is stable for years. On the other hand, immunofluorescence is easier to perform than immunoperoxidase, the conjugated antibody is generally more stable and so more reliable, and the fluorescence signal is easier to demonstrate in black-and-white photography. If a good fluorescence microscope is available and the signal is expected to be reasonably strong, then immunofluorescence is the method of choice.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Karush, F. (1978) The affmity of antibody: Range, variability, and the role of multivalence, in Comprehensive Immunology, vol. 5 Immunoglabuins (Litman, G. W. and Good, R. A., eds.) Plenum, New York and London, pp. 85–116.
Harlow, E. and Lane. D. (1988) Antibadies A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).
Alzari, P. M., Lascombe, M. B., and Poljak, R.J. (1988) Three-dimensional structure of antibodies. Annu. Rev. Immunol 6, 555–580.
Crumpton, M. J. (1974) Protein antigens: The molecular basis of antigenicity and immunogenicity, in The Antigens, vol. 2 (Sela, M., ed.) Academic, New York, pp. 1–78.
Tainer, J. A., Getzoff, E. D., Paterson, Y., Olson, A. J., and Lerner, R. A. (1985) The atomic mobility component of protein antigenicity. Annu. Rev. Immunol. 3, 501–535.
Milstein, C., Wright. B., and Cuello, A. C. (1983) The discrepancy between the crossreactivity of a monoclonal antibody to serotonin and its immunohistochemical specificity. Mol. Immunol 20, 113–123.
Mason, D. W. and Williams, A. F. (1980) The kinetics of antibody binding to membrane antigens in solution and at the cell surface. Biochem.J. 187, 1–20.
Dower, S. K., Titus, J. A., and Segal, D. M. (1984) The binding of multivalent ligands to cell surface receptors, in Cell Surface Dynamics (Perelson, A., ed.) Dekker, New York, pp. 277–328.
Carson, K. A. and Mesulam, M.-M. (1982) Electron microscopic demonstration of neural connections using horseradish peroxidase: A comparison of the tetramethylbenzidine procedure with seven other histochemical methods. J. Histochem. Cytochem.30,425–435.
Trojanowski, J. Q., Obrocka. M. A., and Lee, V. M. Y. (1983) A comparison of eight different chromagen protocols for the demonstration of immunoreactive neurofilaments or glial filaments in rat cerebellum using the peroxidaseantiperoxidase method and monoclonal antibodies. J Hisrochem Cytochem. 31, 1217–1223.
Scopsi, L. and Larsson, L. I. (1986) Increased sensitivity in peroxidase immunocytochemistry. A comparative study of a number of peroxidase visualisation methods employing a model system. Histochemistry 84 221–230.
Mason, D. Y and Sammons, R. (1978) Alkaline phosphatasc and peroxidase for double immunoenzymatic labelling of cellular constituents. J. Clin. Puthol. 31, 454–460.
Wilson, M. B. and Nakane, P. K (1978) Recent developments in the periodate method of conjugating horseradish peroxidase (HRPO) to antibodies, in Immunofluorescence and Related Staining Techniques (Knapp, W., Holubar, K. and Wick, G., eds.) Elsevier/North Holland Biomedical, New York, pp. 215–224.
Eldred, W. D., Zucker, C, Karten, H.J., and Yazulla, S. (1983) Comparison of fixation and penetration enhancement techniques for use in ultrastructural immunocytochemistry. J Histochem. Cytochem. 31, 285–292.
Bohn, W. (1978) A fixation method for improved antibody penetration in electron microscopical immunoperoxidase studies. J. Histochem Cytochem. 26, 293–297.
Willingham, M. C., Yamada, S., and Pastan, I. (1978) Ultrastructural antibody localization of alpha2-macroglobulin in membrane-limited vesicles in cultured cells. Proc. Natl. Acad. Sci. USA 75, 4359–4363.
Morris, R.J. and Barber, P. C. (1983) Fixation of Thy-l in nervous tissue for immunohistochemistry: A quantitative assessment of the effect of different fwation conditions upon retention of antigenicity and the cross-linking of Thy-l. J. Histochem. Cytochem. 31, 263–274.
Willingham, M. C. and Yamada, S. S. (1979) Development of a new primary fxative for electron microscopic immunocytochemical local&&ion of intracellular antigens in cultured cells. J. Histochem. Cytochem. 27, 947–960.
McClean. I. W. and Nakane, P. K (1974) Periodate-lysine-paraformaldehyde fixative, a new fixative for immunoelectron microscopy. J Histochem. Cybchem.22, 1077–1083.
Hickson, D. C., Yep, J. N., Glenney, J. R., Hayes. T., and WaIdborg, E. F. (1981) Evaluation of periodate/lysine/paraformaldehyde fixation as a method for cross-linking plasma membrane glycoproteins. J.Histochem. Cytochem. 29, 561–566.
Weisburger, E. R., Russfield, A. B., Homburger, F., Weisburger, J. H., Boger, E., van Dongen, C. G., and Chu. K. C. (1978) Testing of 21 environmental aromatic amines or derivatives for long-term toxicity or carcinogenicity. J Environ. Pathol. Toxicol 2, 325–356.
Gill, G. W., Frost, J. R., and Miller, K. A. (1974) A new formula for a half-oxidized hematoxylin solution that neither overstains nor requires differentiation. Acta Cyto (Baltimore) 18, 300–311.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1991 The Humana Press Inc., Clifton, NJ
About this protocol
Cite this protocol
Morris, R. (1991). Immunoperoxidase Staining of Gene Products in Cultured Cells Using Monoclonal Antibodies. In: Murray, E.J. (eds) Gene Transfer and Expression Protocols. Methods in Molecular Biology, vol 7. Humana Press. https://doi.org/10.1385/0-89603-178-0:339
Download citation
DOI: https://doi.org/10.1385/0-89603-178-0:339
Publisher Name: Humana Press
Print ISBN: 978-0-89603-178-4
Online ISBN: 978-1-59259-494-8
eBook Packages: Springer Protocols