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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
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2055)

Abstract

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 

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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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