Hapten–Anti-Hapten Technique for Two-Color IHC Detection of Phosphorylated EGFR and H2AX Using Primary Antibodies Raised in the Same Host Species

  • Jodi Hagen
  • David Schwartz
  • Alexander E. Kalyuzhny
Part of the Methods in Molecular Biology book series (MIMB, volume 1554)


Multiplex staining of cell and tissue sections with antibodies raised in the same host species is a serious challenge because of unwanted but inevitable cross-reactivity of secondary antibodies with irrelevant primary antibodies. Several techniques can be used to overcome this obstacle including direct labeling of primary antibodies with fluorescent tags and using tyramide signal amplification. Unfortunately these techniques either lack sensitivity, or require a long multistep protocol which can cause physical damage of specimens. As an alternative, we have developed a protocol based on conjugation of primary antibodies to small-size hapten molecules which can be detected with hapten-specific fluorescent secondary antibodies. This technique has been used for two-color labeling of Y845 phosphorylated Epidermal Growth Factor Receptor (EGFR) and S139 phosphorylated histone H2AX protein in A431 human epidermoid carcinoma cells. Our novel hapten–anti-hapten detection chemistry allows for generating a stronger fluorescent signal and completely avoid cross-interactions of secondary antibodies with irrelevant primary antibodies.

Key words

Two-color immunofluorescence Hapten-conjugated primary antibodies Anti-hapten secondary antibodies Phospho-specific antibodies Phosphorylated EGFR Phosphorylated H2AX 


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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Jodi Hagen
    • 1
  • David Schwartz
    • 2
  • Alexander E. Kalyuzhny
    • 3
  1. 1.Bio-Techne, Inc.MinneapolisUSA
  2. 2.Cell IDx, Inc.San DiegoUSA
  3. 3.Immunocytochemistry & Elispot Assays, Bio-Techne, Inc.MinneapolisUSA

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