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* These authors contributed equally
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.
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.
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....
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.
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.......
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.......
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).
....Name | Company | Catalog Number | Comments |
Antibody diluent | DAKO | REF S3022 | Blocking |
Peroxidase Blocking Solution | DAKO | S2023 | For peroxide blocking |
Vectra multispectral automated microscope | Perkin Elmer | Vectra2.0.8 | Spectral imaging |
absolute Ethanol | EMSURE | 1.00983.2500 | Ethyl alcohol for rehydration |
Amplification Diluent | PERKIN ELMER | FP1135 | Fluorophore diluent buffer |
Anti-Mouse IgG [Goat] HRP-Labeled | PERKIN ELMER | NEF822001EA | Secondary antibody |
Anti-Rabbit IgG [Goat] HRP-Labeled | PERKIN ELMER | NEF812001EA | Secondary antibody |
BCL2 | DAKO | clone 124 ( Lot No. 20031561)(RRID-AB578693) | primary antibody |
BCL6 | LEICA | NCL-L-Bcl6-564(Lot No.6050438)(RRID-AB563429) | primary antibody |
CD20 | DAKO | Clone L26 (Lot No.20028627) (RRID-AB442055) | primary antibody |
c-MYC | ABCAM | Y 69 clone ab32072 (Lot NO.GR29511133)(RRID-AB731658) | primary antibody |
Cy 5 | PERKIN ELMER | FP1171 | Appropriate tyramide based fluorescent reagent |
Graphpad Prism 7 | Graph pad | Statisitcal software | |
HistoClear Clearing Agent | SIGMA | H2779-1L | Histoclear for dewaxing and clearing |
inForm Advanced Image Analysis Software | Perkin Elmer | Inform Software 2.2.1 | Data Analysis software |
KI67 | DAKO | Clone MIB-1 (Lot No.20040401) (RRID-AB2314699) | primary antibody |
KOS MILESTONE multifunctional tissue processor | Milestone | Microwave for Heat induced epitope retrieval | |
Microwave | PANASONIC | NN-ST651M | Microwave stripping |
Mowiol | SIGMA ALDRICH | 81381 Aldrich Mowiol® 4-88 | mounting media |
Opal 570 | PERKIN ELMER | FP1488 | Appropriate tyramide based fluorescent reagent |
Opal520 | PERKIN ELMER | FP1487B21 | Appropriate tyramide based fluorescent reagent |
Opal540 | PERKIN ELMER | FP1494 | Appropriate tyramide based fluorescent reagent |
Opal620 | PERKIN ELMER | FP1495 | Appropriate tyramide based fluorescent reagent |
Poly-L-lysine coated slide | FISHER SCIENTIFIC | 120-550-15 | Slide for tissue section adhesion in routine histological use |
Spectral DAPI | PERKIN ELMER | FP1490 | nucleic acid stain |
Target Retrieval Solution, pH9.0(10x) | DAKO | S2367 | For HIER |
Tris Buffer saline (TBS) | 1st BASE | BUF3030 20X4L | for buffer wash |
Tween 20 | SIGMA ALDRICH | P1379-1L | Tween |
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