Multicolor enzymatic immunohistochemistry assays for FFPE tissue

White paper by Shenna L. Washington, Pamela Y. Johnson, Mary D. Beauchamp, Priya Handa, *Augustine Mzumara (*Corresponding Author)


This white paper was produced in collaboration with the Histology & Imaging Core Laboratory at the Benaroya Research Institute (BRI), Seattle, WA, USA. The BRI Histology Core helps investigators to better understand how cell behavior and gene expression in tissues and organs are affected by disease processes.


While immunofluorescence (IF) has become a preferred method of concurrently detecting multiple antigenic markers within a single tissue specimen, immunoenzymatic chromogen staining with multiple colored substrates remains an informative and important research tool (1, 2). Staining specimens with immunoenzymatic chromogens allows researchers to cast a broader net for investigating targets because, unlike IF, it is permanent and can be visualized in relation to the comprehensive morphology of tissue specimens (1, 2). This stability also allows standard histological stains to be used in conjunction with the immunohistochemistry (IHC) to give researchers an additional layer of information.

Immunofluorescence is often preferred over enzymatic IHC because it is a technically simpler method of visualizing multiple antigenic markers. Imaging with a fluorescent microscope and creating the composite images of multiple IF color channels can be the most complicated aspect of IF staining, but quantification of distinctly stained elements is simple and precise. The development of assays involving multiple IHC chromogenic substrates presents many challenges, such as determining the appropriate sequence of marker application/detection, compatibility of cellular localization of combined markers, special requirements for preparation of various enzymatic substrates, visual contrast compatibility of chromogenic substrates, the length of the overall staining process, and methods of analyzing staining results (1, 2). These potential IHC development obstacles can take time to overcome, but when the IHC assay is complete, the various chromogens can be visualized simultaneously, using standard light microscopy, and can be viewed repeatedly without altering staining results. These qualities of multicolor IHC are of significant value to researchers, especially in the early phases of study.

There are now many tools available to easily resolve some of the significant assay development obstacles of multicolor enzymatic immunohistochemistry. Abcam has developed kits for easy antibody conjugation (both Horseradish Peroxidase and Alkaline Phosphatase), and a range of chromogenic substrates with improved stability. These products reduce the challenges of involved substrate preparation, and significantly reduce the length of staining procedures. The present work discusses how improved reagents simplify multicolor enzymatic IHC assay development for Formalin-Fixed Paraffin-Embedded (FFPE) tissues.

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Benaroya white paper triple stain

Formalin-fixed, paraffin-embedded human spleen tissue (63x) with the combination of co-stained elements: Abcam Active Caspase 3 in DAB (brown), Dako CD68 in alkaline phosphatase red (red/pink), and Abcam Iron Stain with its blue reaction product.


  • 1. Van der Loos, C.M. “Chromogens in Multiple Immunohistochemical Staining Used for Visual Assessment and Spectral Imaging: The Colorful Future”. The Journal of Histotechnology/Vol.33, No. 1/March 2010.
  • 2. Van der Loos, C.M. “Practical Guide to Multiple Staining”. Biotechniques, November 2009. Web. 10 January 2014. Web. 10 January 2014.
  • 3. Van der Loos, C.M. “Multicolor Immunohistochemistry: staining, spectral unmixing, co-localization, quantitation, and beyond”. PerkinElmer, 13 June 2012. Web. 10 January 2014.