The InSituPlex® Staining Method for Multiplexed Immunofluorescence Cell Phenotyping and Spatial Profiling of Tumor FFPE Samples

  • Mael Manesse
  • Katir K. Patel
  • Mark Bobrow
  • Sean R. DowningEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2055)


Multiplexed immunohistochemistry (mIHC) enables the detection, quantification, and localization of many markers within cell or tissue samples, leading to a better understanding of the architecture of a disease at the cellular level. Current mIHC techniques involve long staining and assay times, require dedicated and/or captive instrumentation, and entail tedious assay optimization, hindering their establishment as routine methods. Here, we demonstrate the use of the InSituPlex® method for spatial profiling of immuno-oncology targets in FFPE tumor tissue with the UltiMapper™ I/O PD-L1 multiplex assay. The panel consists of five protein markers to profile immune infiltration and PD-L1 expression and includes CD8, CD68, PD-L1, pan CK, and SOX10 markers. The assay shows benefits of high and low expression of markers, coexpression and colocalization of proteins in single cells, and completion of staining and image acquisition in 5.5 h. Through the combination of multiplexed characterization of protein expression in whole tissue sections, fast staining workflow, and compatibility with existing instrumentation, the InSituPlex method provides a robust modality for deep phenotyping of the tumor and its microenvironment.

Key words

Immunohistochemistry Multiplex immunofluorescence Immune checkpoint Tumor microenvironment 


  1. 1.
    Berthel A, Zoernig I, Valous NA, Kahlert C, Klupp F, Ulrich A et al (2017) Detailed resolution analysis reveals spatial T cell heterogeneity in the invasive margin of colorectal cancer liver metastases associated with improved survival. Oncoimmunology 6(3):e1286436CrossRefGoogle Scholar
  2. 2.
    Gartrell RD, Marks DK, Hart TD, Li G, Davari DR, Wu A et al (2018) Quantitative analysis of immune infiltrates in primary melanoma. Cancer Immunol Res 6(4):481–493CrossRefGoogle Scholar
  3. 3.
    Feng Z, Puri S, Moudgil T, Wood W, Hoyt CC, Wang C et al (2015) Multispectral imaging of formalin-fixed tissue predicts ability to generate tumor-infiltrating lymphocytes from melanoma. J Immunother Cancer 3(1):47CrossRefGoogle Scholar
  4. 4.
    Feng Z, Bethmann D, Kappler M, Ballesteros-Merino C, Eckert A, Bell RB et al (2017) Multiparametric immune profiling in HPV- oral squamous cell cancer. JCI Insight 2(14):93652CrossRefGoogle Scholar
  5. 5.
    Maby P, Tougeron D, Hamieh M, Mlecnik B, Kora H, Bindea G et al (2015) Correlation between density of CD8+ T-cell infiltrate in microsatellite unstable colorectal cancers and frameshift mutations: a rationale for personalized immunotherapy. Cancer Res 75(17):3446–3455CrossRefGoogle Scholar
  6. 6.
    Gerdes MJ, Sevinsky CJ, Sood A, Adak S, Bello MO, Bordwell A et al (2013) Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue. Proc Natl Acad Sci U S A 110(29):11982–11987CrossRefGoogle Scholar
  7. 7.
    Sood A, Miller AM, Brogi E, Sui Y, Armenia J, McDonough E et al (2016) Multiplexed immunofluorescence delineates proteomic cancer cell states associated with metabolism. JCI Insight 1(6)Google Scholar
  8. 8.
    Chattopadhyay PK, Price DA, Harper TF, Betts MR, Yu J, Gostick E et al (2006) Quantum dot semiconductor nanocrystals for immunophenotyping by polychromatic flow cytometry. Nat Med 12(8):972–977CrossRefGoogle Scholar
  9. 9.
    Chattopadhyay PK, Gaylord B, Palmer A, Jiang N, Raven MA, Lewis G et al (2012) Brilliant violet fluorophores: a new class of ultrabright fluorescent compounds for immunofluorescence experiments. Cytom Part A 81A(6):456–466CrossRefGoogle Scholar
  10. 10.
    Rimm DL (2006) What brown cannot do for you. Nat Biotechnol 24(8):914–916CrossRefGoogle Scholar
  11. 11.
    Tóth ZE, Mezey É (2007) Simultaneous visualization of multiple antigens with Tyramide signal amplification using antibodies from the same species. J Histochem Cytochem 55(6):545–554CrossRefGoogle Scholar
  12. 12.
    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(1):46–58CrossRefGoogle Scholar
  13. 13.
    Long DJ, Buggs C, Buggs C (2008) Microwave oven-based technique for immunofluorescent staining of paraffin-embedded tissues. J Mol Histol 39(1):1–4CrossRefGoogle Scholar
  14. 14.
    Buchwalow IB, Minin EA, Boecker W (2005) A multicolor fluorescence immunostaining technique for simultaneous antigen targeting. Acta Histochem 107(2):143–148CrossRefGoogle Scholar
  15. 15.
    Zhang W, Hubbard A, Jones T, Racolta A, Bhaumik S, Cummins N et al (2017) Fully automated 5-plex fluorescent immunohistochemistry with tyramide signal amplification and same species antibodies. Lab Investig 97(7):873–885CrossRefGoogle Scholar
  16. 16.
    Lin J-R, Izar B, Wang S, Yapp C, Mei S, Shah PM et al (2018) Highly multiplexed immunofluorescence imaging of human tissues and tumors using t-CyCIF and conventional optical microscopes. elife 7:e31657CrossRefGoogle Scholar
  17. 17.
    Lin J-R, Fallahi-Sichani M, Sorger PK (2015) Highly multiplexed imaging of single cells using a high-throughput cyclic immunofluorescence method. Nat Commun 6(1):8390CrossRefGoogle Scholar
  18. 18.
    Angelo M, Bendall SC, Finck R, Hale MB, Hitzman C, Borowsky AD et al (2014) Multiplexed ion beam imaging of human breast tumors. Nat Med 20(4):436–442CrossRefGoogle Scholar
  19. 19.
    Levenson RM, Borowsky AD, Angelo M (2015) Immunohistochemistry and mass spectrometry for highly multiplexed cellular molecular imaging. Lab Investig 95(4):397–405CrossRefGoogle Scholar
  20. 20.
    Giesen C, Wang HAO, Schapiro D, Zivanovic N, Jacobs A, Hattendorf B et al (2014) Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry. Nat Methods 11(4):417–422CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Mael Manesse
    • 1
  • Katir K. Patel
    • 1
  • Mark Bobrow
    • 1
  • Sean R. Downing
    • 1
    Email author
  1. 1.UltivueCambridgeUSA

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