Abstract
The use of specific antibodies labeled with a fluorescent dye to localize substances in tissues was first devised by A. H. Coons and his associates. At first, the specific antibody itself was labeled and applied to the tissue section to identify the antigenic sites (direct method) (1). Later, the more sensitive and versatile indirect method (2) was introduced. The primary, unlabeled, antibody is applied to the tissue section, and the excess is washed off with buffer. A second, labeled antibody from another species, raised against the IgG of the animal donating the first antibody, is then applied. The primary antigenic site is thus revealed. A major advantage of the indirect method is the enhanced sensitivity. In addition, a labeled secondary antibody can be used to locate any number of primary antibodies raised in the same animal species without the necessity of labeling each primary antibody.
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References
Coons, A. H., Creech, H. J., and Jones, R. N. (1941) Immunological properties of an antibody containing a fluorescent group. Proc. Soc. Exp. Biol. Med. 47, 200–202.
Coons, A. H., Leduc, E. H., and Connolly, J. M. (1955) Studies on antibody production. I. A method for the histochemical demonstration of specific antibody and its application to a study of the hyperimmune rabbit. J. Exp. Med. 102, 49–60.
Titus, J. A., Haugland, R., Sharrow, S. O., and Segal, D. M. (1982) Texas Red, a hydrophilic, red-emitting fluorophore for use with fluorescein in dual parameter flow microfluorometric and fluorescence microscopic studies. J. Immunol. Methods 50, 193–204.
Oi, V. T., Glazer, A. N., and Stryer, L. (1982) Fluorescent phycobiliprotein conjugates for analyses of cells and molecules. J. Cell Biol. 93, 981–986.
Bochner, B. S., McKelvey, A. A., Schleimer, R. P., Hildreth, J. E., and Mac-Glashan, D. W., Jr. (1989) Flow cytometric methods for the analysis of human basophil surface antigens and viability. J. Immunol. Methods 125, 265–271.
Schreiber, A. B. and Haimovich, J. (1983) Quantitative fluorometric assay for detection and characterization of Fc receptors. Methods Enzymol. 93, 147–155.
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© 1999 Humana Press Inc., Totowa, NJ
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Mao, SY. (1999). Conjugation of Fluorochromes to Antibodies. In: Javois, L.C. (eds) Immunocytochemical Methods and Protocols. Methods in Molecular Biology™, vol 115. Humana Press. https://doi.org/10.1385/1-59259-213-9:35
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DOI: https://doi.org/10.1385/1-59259-213-9:35
Publisher Name: Humana Press
Print ISBN: 978-0-89603-813-4
Online ISBN: 978-1-59259-213-5
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