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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here we describe a protocol for multiplex fluorescent immunohistochemical staining and imaging for the simultaneous localization of multiple cancer-associated antigens in lymphoma. This protocol can be extended to the colocalization analysis of biomarkers within all tissue sections.

Abstract

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic purposes. However, the visualization and quantification of multiple antigens in a single tissue section using conventional chromogenic IHC is challenging. Multiplexed imaging is especially relevant in lymphoma research and diagnostics, where markers have to be interpreted in the context of a complex tumor microenvironment. Here we describe a protocol for multiplexed fluorescent IHC staining to enable the quantitative assessment of multiple targets in specific cell types of interest in lymphoma.The method covers aspects of antibody validation, antibody optimization, the multiplex optimization with markers of lymphoma subtypes, the staining of tissue microarray (TMA) slides, and the scanning of the slides, followed by data analysis, with specific reference to lymphoma. Using this method, scores for both the mean intensity of a marker of interest and the percentage positivity are generated to facilitate further quantitative analysis. Multiplexing minimizes sample utilization and provides spatial information for each marker of interest.

Introduction

Lymphoid neoplasms are caused by the uncontrolled malignant proliferation of lymphocytes. These cells are vital components of the immune system and localize to the primary and secondary immune organs, such as the bone marrow, lymph nodes, spleen, and other mucosa-associated lymphoid system. Lymphoid neoplasms are a heterogeneous group of disorders who are classified based on a constellation of features, including morphology, immunophenotype, genetic features, and clinical presentation. While each parameter plays a part, lineage remains a defining feature and forms the basis for the WHO classification system which recognizes neoplasms derived from B cells, T cells, and....

Protocol

All tissues used in this protocol were obtained under the Singapore NHG Domain Specific Review Board B study 2014/00693.

1. Selection and Validation of Antibodies

NOTE: Before proceeding with the establishment of any multiplexed panel, ensure that all antibodies stain robustly, identifying only the target antigen of interest. The aim is to select antibodies that specifically recognize the antigen of interest in tissue sections.

  1. For an antibody with a wel.......

Representative Results

mf-IHC images for a DLBCL sample with C-MYC and BCL2 gene rearrangement (double-hit lymphoma) are shown in Figure 1. Figure 2 illustrates the simulated bright-field immunohistochemical images. Figure 3 indicates the generation of percentage data. Figure 4 displays the details of a median formula for the generation of numeric data. Figure 5 s.......

Discussion

mf-IHC has the potential to enable pathologists to refine diagnosticcriteria in lymphoid pathology and to analyze the role of biomarkers in specific cell types toward a prediction of clinical outcome. As a new research method, mf-IHC is increasingly applied to the quantitative and spatial identification of multiple immune parameters of tumor cells17. The detection of mf-IHC for the co-expression of tumor biomarkers has been shown to be reproducible and reliable5. However, t.......

Acknowledgements

S.-B.N. and A.D.J. are supported by the Singapore Ministry of Health's National Medical Research Council Transition Awards (NMRC/TA/0020/2013 and NMRC/TA/0052/2016). The authors acknowledge a Yong Siew Yoon Research Grant to A.D.J. from the National University Cancer Institute of Singapore toward the purchase of a Vectra spectral imaging microscope. This study is approved by the Singapore NHG Domain Specific Review Board B (2014/00693).

....

Materials

NameCompanyCatalog NumberComments
Antibody diluentDAKOREF S3022Blocking
Peroxidase Blocking SolutionDAKOS2023For peroxide blocking
Vectra multispectral automated microscopePerkin ElmerVectra2.0.8Spectral imaging
absolute EthanolEMSURE1.00983.2500Ethyl alcohol for rehydration
Amplification DiluentPERKIN ELMERFP1135Fluorophore diluent buffer
Anti-Mouse IgG [Goat] HRP-LabeledPERKIN ELMERNEF822001EASecondary antibody
Anti-Rabbit IgG [Goat] HRP-LabeledPERKIN ELMERNEF812001EASecondary antibody
BCL2DAKOclone 124 ( Lot No. 20031561)(RRID-AB578693)primary antibody
BCL6LEICANCL-L-Bcl6-564(Lot No.6050438)(RRID-AB563429)primary antibody
CD20DAKOClone L26 (Lot No.20028627) (RRID-AB442055)primary antibody
c-MYCABCAMY 69 clone ab32072 (Lot NO.GR29511133)(RRID-AB731658)primary antibody
Cy 5PERKIN ELMERFP1171Appropriate tyramide based fluorescent reagent
Graphpad Prism 7Graph padStatisitcal software
HistoClear Clearing AgentSIGMAH2779-1LHistoclear for dewaxing and clearing
inForm Advanced
Image Analysis Software
Perkin ElmerInform Software 2.2.1Data Analysis software
KI67DAKOClone MIB-1 (Lot No.20040401) (RRID-AB2314699)primary antibody
KOS MILESTONE multifunctional tissue processorMilestoneMicrowave for Heat induced epitope retrieval
MicrowavePANASONICNN-ST651MMicrowave stripping
MowiolSIGMA ALDRICH81381 Aldrich Mowiol® 4-88mounting media
Opal 570PERKIN ELMERFP1488Appropriate tyramide based fluorescent reagent
Opal520PERKIN ELMERFP1487B21Appropriate tyramide based fluorescent reagent
Opal540PERKIN ELMERFP1494Appropriate tyramide based fluorescent reagent
Opal620PERKIN ELMERFP1495Appropriate tyramide based fluorescent reagent
Poly-L-lysine coated slideFISHER SCIENTIFIC120-550-15Slide for tissue section adhesion in routine histological use
Spectral DAPIPERKIN ELMERFP1490nucleic acid stain
Target Retrieval Solution, pH9.0(10x)DAKOS2367For HIER
Tris Buffer saline (TBS)1st BASEBUF3030 20X4Lfor buffer wash
Tween 20SIGMA ALDRICHP1379-1LTween

References

  1. Swerdlow, S. H., et al. . WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. , (2017).
  2. Swerdlow, S. H., et al. The 2016 revision of the World Health Organization classification of lymphoid neoplasms. Blood. 12....

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