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Multiple Antigen Immunostaining Procedures

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Immunocytochemical Methods and Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 588))

Abstract

Detection of multiple antigens in the same tissue section can be done by combining a range of immunohisto/cytochemical techniques based either on light microscopic chromogenic precipitates or fluorochrome labeling. Light microscopic techniques preferred for this purpose use combinations of immunogold silver staining (black precipitate), immunoperoxidase, immunoalkaline phosphatase and immunogalactosidase methods using chromogens of different colors. Fluorochrome labels favored for these combinations include AMCA (blue), FITC (green), rhodamine (orange-red) and Cy5 (far red), their matching synthetic members from the Alexa series, or quantum dots. Antibodies directly labeled or those from noncross-reacting animal species (e.g., mouse, rabbit, goat, guinea pig etc.) can be applied simultaneously. When the antigens of interest are in separate cells or cell compartments (e.g., in cell membrane, cytoplasm or nucleus), and only cross-reacting antibodies are available, there have also been ways of avoiding unwanted cross-talk. These include the exploitation of the shielding effect of chromogens; inactivation of immuno-sequences of the first staining by using either acidic elution, formaldehyde fixation or microwave heating; combining unlabeled and hapten-labeled antibodies; or using labeled monovalent F(ab) secondary antibodies. In this chapter we briefly discuss the principle of multiple antigen immunolabeling and provide useful protocols for its performance.

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Author information

Authors and Affiliations

  1. 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary

    Tibor Krenacs

  2. Laboratory of Tumor Pathology and Molecular Diagnostics, Bay Zoltan Foundation for Applied Research, Szeged, Hungary

    Laszlo Krenacs

  3. Laboratory of Pathology, Hematopathology Section, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

    Mark Raffeld

Authors
  1. Tibor Krenacs
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  2. Laszlo Krenacs
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  3. Mark Raffeld
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Corresponding author

Correspondence to Tibor Krenacs .

Editor information

Editors and Affiliations

  1. Fac. Medicina de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes 3900, Ribeirão Preto, 14049-900, Brazil

    Constance Oliver

  2. Fac. Medicina de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes 3900, Ribeirão Preto, 14049-900, Brazil

    Maria Célia Jamur

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© 2010 Humana Press, a part of Springer Science+Business Media, LLC

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Krenacs, T., Krenacs, L., Raffeld, M. (2010). Multiple Antigen Immunostaining Procedures. In: Oliver, C., Jamur, M. (eds) Immunocytochemical Methods and Protocols. Methods in Molecular Biology, vol 588. Humana Press. https://doi.org/10.1007/978-1-59745-324-0_28

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  • DOI: https://doi.org/10.1007/978-1-59745-324-0_28

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-463-0

  • Online ISBN: 978-1-59745-324-0

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