Multiplexed Immunohistochemical Consecutive Staining on Single Slide (MICSSS): Multiplexed Chromogenic IHC Assay for High-Dimensional Tissue Analysis

  • Guray Akturk
  • Robert Sweeney
  • Romain Remark
  • Miriam Merad
  • Sacha GnjaticEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2055)


Disease states and cellular compartments can display a remarkable amount of heterogeneity, and truly appreciating this heterogeneity requires the ability to detect and probe each subpopulation present. A myriad of recent single-cell assays has allowed for in-depth analysis of these diverse cellular populations; however, fully understanding the interplay between each cell type requires knowledge not only of their mere presence but also of their spatial organization and their relation one to the other. Immunohistochemistry allows for the visualization of cells and tissue; however, standard techniques only allow for the use of very few probes on a single specimen, not allowing for in-depth analysis of complex cellular heterogeneity. A number of multiplex imaging techniques, such as immunofluorescence and multiplex immunohistochemistry, have been proposed to allow probing more cellular markers at once; however, many of these techniques still have their limitations. The use of fluorescent markers has an inherent limitation to the number of probes that can be simultaneously used due to spectral overlap. Moreover, other proposed multiplex IHC methods are time-consuming and require expensive reagents. Still, many of the methods rely on frozen tissue, which deviates from standards in human pathological evaluation. Here, we describe a multiplex IHC technique, staining for consecutive markers on a single slide, which utilizes similar steps and similar reagents as standard IHC, thus making it possible for any lab with standard IHC capabilities to perform this useful procedure. This method has been validated and confirmed that consecutive markers can be stained without the risk of cross-reactivity between staining cycles. Furthermore, we have validated that this technique does not lead to decreased antigenicity of subsequent epitopes probed, nor does it lead to steric hindrance.

Key words

Multiplexed immunohistochemistry Chromogenic immunohistochemistry Immunostaining Whole slide imaging Consecutive staining Serial staining Single slide Image analysis Positive cell detection Histology Morphology Cell segmentation Machine learning Random forest Cancer immunotherapy Immuno-oncology Biomarkers In situ markers PD-L1 PD-1 CD3 CD8 FOXP3 CD20 CD66b CD68 


  1. 1.
    Galon J et al (2012) Cancer classification using the Immunoscore: a worldwide task force. J Transl Med 10:205CrossRefGoogle Scholar
  2. 2.
    Remark R et al (2013) Characteristics and clinical impacts of the immune environments in colorectal and renal cell carcinoma lung metastases: influence of tumor origin. Clin Cancer Res 19:4079–4091CrossRefGoogle Scholar
  3. 3.
    Pardoll DM (2012) The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer 12:252–264CrossRefGoogle Scholar
  4. 4.
    Page DB, Postow MA, Callahan MK, Allison JP, Wolchok JD (2014) Immune modulation in cancer with antibodies. Annu Rev Med 65:185–202CrossRefGoogle Scholar
  5. 5.
    Spitzer MH, Nolan GP (2016) Mass cytometry: single cells, many features. Cell 165:780–791CrossRefGoogle Scholar
  6. 6.
    Robertson D, Savage K, Reis-Filho JS, Isacke CM (2008) Multiple immunofluorescence labelling of formalin-fixed paraffin-embedded (FFPE) tissue. BMC Cell Biol 9:13–10CrossRefGoogle Scholar
  7. 7.
    Giesen C et al (2014) Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry. Nat Methods 11:417–422CrossRefGoogle Scholar
  8. 8.
    Gerdes MJ et al (2013) Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue. Proc Natl Acad Sci U S A 110:11982–11987CrossRefGoogle Scholar
  9. 9.
    Stack EC, Wang C, Roman KA, Hoyt CC (2014) Multiplexed immunohistochemistry, imaging, and quantitation: a review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis. Methods 70:46–58CrossRefGoogle Scholar
  10. 10.
    Angelo M et al (2014) Multiplexed ion beam imaging of human breast tumors. Nat Med 20:436–442CrossRefGoogle Scholar
  11. 11.
    Remark R et al (2016) In-depth tissue profiling using multiplexed immunohistochemical consecutive staining on single slide. Sci Immunol 1:aaf6925–aaf6925CrossRefGoogle Scholar
  12. 12.
    Bankhead P et al (2017) QuPath: open source software for digital pathology image analysis. Sci Rep 7:16878CrossRefGoogle Scholar
  13. 13.
    Rueden, C. T.; Schindelin, J. & Hiner, M. C. et al. (2017), “ImageJ2: ImageJ for the next generation of scientific image data”, BMC Bioinformatics 18:529, PMID 22743772, doi:10.1038/nmeth.2019CrossRefGoogle Scholar
  14. 14.
    Albert Cardona, Stephan Saalfeld, Johannes Schindelin, Ignacio Arganda-Carreras, Stephan Preibisch, Mark Longair, Pavel Tomancak, Volker Hartenstein and Rodney J. Douglas. 2012. TrakEM2 Software for Neural Circuit Reconstruction. PLoS ONE 7(6): e38011.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Guray Akturk
    • 1
  • Robert Sweeney
    • 1
  • Romain Remark
    • 2
  • Miriam Merad
    • 1
  • Sacha Gnjatic
    • 1
    Email author
  1. 1.Tisch Cancer InstituteIcahn School of Medicine at Mount SinaiNew YorkUSA
  2. 2.Innate PharmaMarseilleFrance

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