Double Immunohistochemistry and Digital Image Analysis

  • Pablo Moreno-RuizEmail author
  • Lina Wik Leiss
  • Artur Mezheyeuski
  • Monika Ehnman
Part of the Methods in Molecular Biology book series (MIMB, volume 1913)


Immunohistochemistry (IHC) is a commonly used technique for protein detection in tissue sections. The method requires high-affinity antibodies that are specific for the target proteins of interest. More advanced IHC techniques have been developed to meet the need for simultaneous detection of more than one target protein in the same tissue section. This chapter provides general guidelines for double IHC staining of formalin-fixed, paraffin-embedded tissue sections. Chromogenic substrates are chosen based on their excellent contrast and compatibility with the subsequent digital image analysis.

Key words

FFPE Double immunohistochemistry Antibody Chromogenic substrate Digital image analysis 



This work was supported by the Svenska Institutet to AM, the Swedish Childhood Cancer Fund (Barncancerfonden) and the Tornspiran Foundation (Stiftelsen Tornspiran) to ME, and the Knut and Alice Wallenberg Foundation to PM.


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Pablo Moreno-Ruiz
    • 1
    Email author
  • Lina Wik Leiss
    • 1
    • 2
  • Artur Mezheyeuski
    • 3
  • Monika Ehnman
    • 1
  1. 1.Department of Oncology-PathologyKarolinska InstitutetStockholmSweden
  2. 2.Neuro ClinicHaukeland University HospitalBergenNorway
  3. 3.Department of Immunology, Genetics and PathologyUppsala UniversityUppsalaSweden

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