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Cancer Research

Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens

Published: January 21st, 2019



1Laboratory of Pathology, MedImmune, 2Laboratory of Pathology, MedImmune, 3Akoya Biosciences Inc., 4Department of Translational Molecular Pathology, University of Texas - MD Anderson Cancer Center

A detailed protocol for a six-marker multiplex immunofluorescence panel is optimized and performed, using an automated stainer for more consistent results and a shorter procedure time. This approach can be directly adapted by any laboratory for immuno-oncology studies.

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of tissue samples from formalin-fixed, paraffin-embedded (FFPE) biopsies has become a key tool for understanding the complexity of tumor immunology and discovering novel predictive biomarkers for cancer immunotherapy. Immunoprofiling analysis of tissues requires the evaluation of combined markers, including inflammatory cell subpopulations and immune checkpoints, in the tumor microenvironment. The advent of novel multiplex immunohistochemical methods allows for a more efficient multiparametric analysis of single tissue sections than does standard monoplex immunohistochemistry (IHC). One commercially available multiplex immunofluorescence (IF) method is based on tyramide-signal amplification and, combined with multispectral microscopic analysis, allows for a better signal separation of diverse markers in tissue. This methodology is compatible with the use of unconjugated primary antibodies that have been optimized for standard IHC on FFPE tissue samples. Herein we describe in detail an automated protocol that allows multiplex IF labeling of carcinoma tissue samples with a six-marker multiplex antibody panel comprising PD-L1, PD-1, CD68, CD8, Ki-67, and AE1/AE3 cytokeratins with 4′,6-diamidino-2-phenylindole as a nuclear cell counterstain. The multiplex panel protocol is optimized in an automated IHC stainer for a staining time that is shorter than that of the manual protocol and can be directly applied and adapted by any laboratory investigator for immuno-oncology studies on human FFPE tissue samples. Also described are several controls and tools, including a drop-control method for fine quality control of a new multiplex IF panel, that are useful for the optimization and validation of the technique.

Immunoprofiling analysis of FFPE tumor tissue samples has become an essential component of immuno-oncology studies, particularly for the discovery and validation of novel predictive biomarkers for cancer immunotherapy in the context of clinical trials1,2. Chromogenic IHC, using chemical chromogens such as diaminobenzidine, remains the standard technique in diagnostic pathology for the immunolabeling of biopsy tissue3. Standard IHC can also be used for cancer tissue immunoprofiling, including the quantitation of subpopulations of tumor-associated lymphocytes and the assessment of express....

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NOTE: The protocol presented here describes how to perform immunoprofiling of an mIF panel by using TSA for six antibodies (CD68, ki67, PD-L1, PD-1, CD8, and AE1/AE3) on an automated stainer (see Table of Materials). The protocol also describes how to perform the drop controls for a quality control of a new mIF panel (see Supplemental Materials). In this protocol, staining is performed with eight unstained FFPE slides from human tonsil (positive control) and eight unstai.......

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The protocol described here will provide results like those shown in Figure 2. Start with an evaluation of the staining in the tonsil control, beginning with the surface squamous cell epithelium. The histology of the tonsil sample can be reviewed with a pathologist, using the H&E slide as a reference. If chromogenic IHC sections are performed with the same markers on the same tissue block, then these can be used to confirm the density and distribution of .......

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The ongoing cancer immunotherapy revolution is opening novel and promising therapeutic options for cancer patients13. Advances in the field of immuno-oncology will require increased knowledge of the inflammatory tumor microenvironment, not only to understand the biology of the immunological mechanisms involved in carcinogenesis but also to find predictive biomarkers for new immunotherapy-based treatments1,2. Due to the complex biology of c.......

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Editorial support was provided by Deborah Shuman of MedImmune.


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Name Company Catalog Number Comments
"InForm 2.4.2" Software for Spectral Unmixing and Image Analysis PerkinElmer CLS151066 Called "spectral unmixing software" in text
"Phenochart 1.0.9" QPTIFF Software for Selection of MSI and Overall Slide Scan Viewing PerkinElmer CLS151067 Called "QPTIFF software" in text
#1.5 Coverslips Sigma Aldrich 2975246
200 Proof Ethanol Koptec V1001
20x Tris-Buffered Saline VWR J640-4L
Antibody Diluent DAKO S2203
Anti-CD68 Mouse Monoclonal DAKO M087601-2 Clone PG-M1
Anti-CD8 Rabbit Monoclonal Ventana M5392 Clone SP239
Anti-CK Mouse Monoclonal DAKO M351501-2 Clone AE1/AE3
Anti-ki67 Mouse Monoclonal DAKO M724001-2 Clone MIB-1
Anti-PD-1 Rabbit Monoclonal Cell Signaling #86163 Clone D4W2J
Anti-PD-L1 Rabbit Monoclonal Ventana 790-4905 Clone SP263
Bond Dewax Solution Leica AR9222 Called "dewax solution" in text
Bond Epitope Retrieval Solution 1 Leica AR9961 Called "ER1" in text
Bond Epitope Retrieval Solution 2 Leica AR9640 Called "ER2" in text
Bond Open Containers, 30 mL Leica OP309700 Called "30 mL open containers" in text
Bond Open Containers, 7 mL Leica OP79193 Called "7 mL open containers" in text
Bond Polymer Refine Detection Leica DS9800 Called "chromogenic detection kit" in text
Bond Research Detection Kit Leica DS9455 Called "research detection kit" in text
Bond Titration Kit Leica OPT9049 Called "titration kit" in text
Bond Universal Covertile Novocastra Leica S21.2001 Called "covertiles" in text
Bond Wash Solution 10X Concentrate Leica AR9590 Called "10x wash solution" in text
BondRX Autostainer Leica Called "automated stainer" in text
BondRX Software Version Leica Called "automated stainer software" in text
Opal 7-Color Automation IHC Kit PerkinElmer NEL801001KT Called "multispectral staining kit" in text
Peroxidase Block Leica RE7101
ProLong Diamond Antifade Mountant Thermo P36965 Called "slide mountant" in text
Starfrost Slides Fisher 15-183-51
Vectra Polaris Multispectral Microscope with "Vectra 3.0.5" Software for Multispectral Microscope Control PerkinElmer CLS143455 Called "microscope control software" in text

  1. Bethmann, D., Feng, Z., Fox, B. A. Immunoprofiling as a predictor of patient's response to cancer therapy-promises and challenges. Current Opinion in Immunology. 45, 60-72 (2017).
  2. Taube, J. M., et al. Implications of the tumor immune microenvironment for staging and therapeutics. Modern Pathology. 31 (2), 214-234 (2018).
  3. Idikio, H. A. Immunohistochemistry in diagnostic surgical pathology: contributions of protein life-cycle, use of evidence-based methods and data normalization on interpretation of immunohistochemical stains. International Journal of Clinical and Experimental Pathology. 3 (2), 169-176 (2009).
  4. Rebelatto, M. C., et al. Development of a programmed cell death ligand-1 immunohistochemical assay validated for analysis of non-small cell lung cancer and head and neck squamous cell carcinoma. Diagnostic Pathology. 11 (1), 95 (2016).
  5. Parra, E. R., et al. Immunohistochemical and image analysis-based study shows that several immune checkpoints are co-expressed in non-small cell lung carcinoma tumors. Journal of Thoracic Oncology. 13 (6), 779-791 (2018).
  6. Rehman, J. A., et al. Quantitative and pathologist-read comparison of the heterogeneity of programmed death-ligand 1 (PD-L1) expression in non-small cell lung cancer. Modern Pathology. 30 (3), 340-349 (2017).
  7. Dixon, A. R., et al. Recent developments in multiplexing techniques for immunohistochemistry. Expert Review of Molecular Diagnostics. 15 (9), 1171-1186 (2015).
  8. Stack, E. C., Wang, C., Roman, K. A., Hoyt, C. C. Multiplexed immunohistochemistry, imaging, and quantitation: a review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis. Methods. 70 (1), 46-58 (2014).
  9. Feng, Z., et al. Multiparametric immune profiling in HPV- oral squamous cell cancer. JCI Insight. 2 (14), (2017).
  10. Feng, Z., et al. Multispectral imaging of formalin-fixed tissue predicts ability to generate tumor-infiltrating lymphocytes from melanoma. Journal for ImmunoTherapy of Cancer. 3, 47 (2015).
  11. Granier, C., et al. Multiplexed immunofluorescence analysis and quantification of intratumoral PD-1+ Tim-3+ CD8+ T cells. Journal of Visualized Experiments. (132), e56606 (2018).
  12. Parra, E. R., et al. Validation of multiplex immunofluorescence panels using multispectral microscopy for immune-profiling of formalin-fixed and paraffin-embedded human tumor tissues. Scientific Reports. 7 (1), 13380 (2017).
  13. Ribas, A., Wolchok, J. D. Cancer immunotherapy using checkpoint blockade. Science. 359 (6382), 1350-1355 (2018).
  14. Gorris, M. A. J., et al. Eight-color multiplex immunohistochemistry for simultaneous detection of multiple immune checkpoint molecules within the tumor microenvironment. Journal of Immunology. 200 (1), 347-354 (2018).
  15. Parra, E. R., et al. Effect of neoadjuvant chemotherapy on the immune microenvironment in non-small cell lung carcinomas as determined by multiplex immunofluorescence and image analysis approaches. Journal for ImmunoTherapy of Cancer. 6 (1), 48 (2018).
  16. Rimm, D., Schalper, K., Pusztai, L. Unvalidated antibodies and misleading results. Breast Cancer Research and Treatment. 147 (2), 457-458 (2014).
  17. Baskin, D. G., Hewitt, S. M. Improving the state of the science of immunohistochemistry: the Histochemical Society's standards of practice. Journal of Histochemistry & Cytochemistry. 62 (10), 691-692 (2014).
  18. Freedman, L. P., et al. The need for improved education and training in research antibody usage and validation practices. Biotechniques. 61 (1), 16-18 (2016).
  19. Hewitt, S. M. Reproducibility: it is just good science. Journal of Histochemistry & Cytochemistry. 64 (4), 223 (2016).
  20. Uhlen, M., et al. A proposal for validation of antibodies. Nature Methods. 13 (10), 823-827 (2016).

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