Double Immunocytochemical Staining Methods

  • H. Sako
  • Y. Nakane
  • K. Okino
  • K. Nishihara
  • M. Kodama
  • H. Yamada
  • M. Kawata


The introduction of immunological techniques into tissue staining methods has enabled the analysis of the pathological state of various diseases. Immunohistochemical methods are based on the specific antigen-antibody reaction and have made possible the differentiation of the fine structure that could not be stained by ordinary histological staining techniques hitherto. For example, two kinds of cells difficult to differentiate by light microscopy can be visualized separately by using immunohistochemistry in conjuction with different antisera. Furthermore, simultaneous visualization of two or more antigens in the same specimen has become desirable in immunohistochemistry for the detection of cytoarchitectural relationships between cellular sources of these antigens and for quantitating cells containing different antigens (Hsu and Soban, 1982). Therefore, many studies on double staining methods have been carried out.


Double Staining Immunoperoxidase Method Normal Mouse Serum Antibody Method Human Lymph Node 
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  1. Clement, L. T., Dagg, M. K. and Landay, A. (1984). Characterization of human lymphocyte subpopulations: alloreactive cytotoxic T-lymphocyte precursor and effector cells are phenotypically distinct from Leu 2+ suppressor cells. J. Clin. Immunol. 4, 395–402.CrossRefPubMedGoogle Scholar
  2. Coons, A. H. (1958) Fluorescent antibody method. In Danilli, J. F. (ed.), General Cytochemical Methods, Vol. I, Academic Press, New York, pp. 399–422.Google Scholar
  3. 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 rabbits. J. Exp. Med. 102, 49–60.PubMedCentralCrossRefPubMedGoogle Scholar
  4. Falini, B., Tabilio, A., Zuccaccia, M. and Martelli, M. F. (1980). Protein A-peroxidase conjugates for two-stage immunoenzyme staining of intracellular antigens in paraffin-embedded tissues. J. Immunol. Methods 39, 111–120.CrossRefPubMedGoogle Scholar
  5. Guesdon, J-L., Ternynck, T. and Avrameas, S. (1979). The use of avidin-biotin interaction in immunoenzymatic techniques. J. Histochem. Cytochem. 27, 1131–1139.CrossRefPubMedGoogle Scholar
  6. Holgate, C. S., Jackson, P., Cowen, P. N. and Bird, C. C. (1983). Immunogold-silver staining: new method of immunostaining with enhanced sensitivity. J. Histochem. Cytochem. 31, 938–944.CrossRefPubMedGoogle Scholar
  7. Hsu, S. M., Raine, L. and Fanger, H. (1981) Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabelled antibody (PAP) procedures. J. Histochem. Cytochem. 29, 577–580.CrossRefPubMedGoogle Scholar
  8. Hsu, S. M. and Soban, E. (1982). Color modification of diaminobenzidine (DAB) precipitation by metallic ions and its application for double immunohistochemistry. J. Histochem. Cytochem. 30, 1079–1082.CrossRefPubMedGoogle Scholar
  9. Köhler, G. and Milstein, C. (1975). Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–497.CrossRefPubMedGoogle Scholar
  10. Landay, A., Gartland, G. L. and Clement, L. T. (1983). Characterization of aphenotypically distinct subpopulation of Leu 2+ cells that suppresses T cell proliferative responses. J. Immunol. 131, 2757–2761.PubMedGoogle Scholar
  11. Lanier, L. L., Engleman, E. G., Gatenby, P., Babcock, G. F., Warner, N. L. and Herzenberg, L. A. (1983). Correlation of functional properties of human lymphoid cell subsets and surface marker phenotypes using multiparameter analysis and flow cytometry. Immunol. Rev. 74, 143–160.CrossRefPubMedGoogle Scholar
  12. Liposits, Zs., Sherman, D., Phelix, C. and Paul, W. K. (1986). A combined light and electron microscopic immunocytochemical method for the simultaneous localization of multiple tissue antigens. Tyrosine hydroxylase immunoreactive innervation of corticotropin releasing factor synthesizing neurons in the paraventricular nucleus of the rat. Histochemistry 85, 95–106.CrossRefPubMedGoogle Scholar
  13. Nakane, P. K. (1968) Simultaneous localization of multiple tissue antigens using the peroxidase-labelled antibody method: a study on pituitary glands of the rat. J. Histochem. Cytochem. 16, 557–560.CrossRefPubMedGoogle Scholar
  14. Nakane, P. K. and Pierce, G. G. (1966). Enzyme-labelled antibodies: preparation and application for localization of antigens. J. Histochem. Cytochem. 14, 929–931.CrossRefPubMedGoogle Scholar
  15. Oi, V. T., Glazer, A. N. and Stryer, L. (1982). Fluorescent phycobiliprotein conjugates for analysis of cells and molecules. J. Cell Biol. 93, 981–986.CrossRefPubMedGoogle Scholar
  16. Okabayashi, T. (1987). Immunohistohemical study on the regional lymph nodes of gastric cancer. Nippon Geka Gakkai Zasshi 88, 529–542.PubMedGoogle Scholar
  17. Pizzolo, G., Chilosi, M. and Chem, D. (1984). Double immunostaining of lymph node sections by monoclonal antibodies using phycoerythrin labelling and haptenated reagents. Am. J. Clin. Pathol. 82, 44–47.PubMedGoogle Scholar
  18. Roth, J. (1982). Applications of immunocolloids in light microscopy: preparation of protein A-silver and protein A-gold complexes and their application for localization of single and multiple antigens in paraffin sections. J. Histochem. Cytochem. 30, 691–696.CrossRefPubMedGoogle Scholar
  19. Sako, H., Nakane, Y., Okino, K., Nishihara, K., Kodoma, M., Kawata, M. and Yamada, H. (1986). Simultaneous detection of B-cells and T-cells by a double immunohistochemical technique using immunogold-silver staining and the avidin-biotin-peroxidase complex method. Histochemistry 86, 1–4.CrossRefPubMedGoogle Scholar
  20. Scopsi, L. and Larsson, L. I. (1985). Increased sensitivity in immunocytochemistry: effects of double application of antibodies and of silver intensification on immunogold and peroxidase-antiperoxidase staining techniques. Histochemistry 82, 321–329.CrossRefPubMedGoogle Scholar
  21. Sternberger, L. A., Hardy, P. H. Jr., Cuculi, J. J. and Meyer, H. G. (1970). The unlabelled antibody-enzyme method of immunohistochemistry: preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirocheters. J. Histochem. Cytochem. 18, 315–333.CrossRefPubMedGoogle Scholar
  22. Sternberger, L. A. and Joseph, S. A. (1979) The unlabelled antibody method: contrasting colour staining of paired pituitary hormones without antibody removal. J. Histochem. Cytochem. 27, 1424–1429.CrossRefPubMedGoogle Scholar
  23. Takeda, I., Horimi, T., Okabayashi, T., Nagae, S., Mukai, K., Kaneda, M., Shimoyama, H., Chono, S., Tokuda, N., Matsuno, T. and Orita, K. (1986). Immunohistological analysis on mechanism of rejection in human renal allografts: functional analysis of infiltrating mononuclear cells using double staining method. Japanese J. Transplantation 21, 509–514.Google Scholar
  24. Tramu, G., Pilley, A. and Leonardelli, J. (1978). An efficient method of antibody elution for the successive or simultaneous localization of two antigens by immunocytochemistry. J. Histochem. Cytochem. 26, 322–324.CrossRefPubMedGoogle Scholar

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© The Editor and Contributors 1989

Authors and Affiliations

  • H. Sako
  • Y. Nakane
  • K. Okino
  • K. Nishihara
  • M. Kodama
  • H. Yamada
  • M. Kawata

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