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Abstract

Immunology and Infection

Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma

Published: January 9th, 2019

DOI:

10.3791/58711

1Department of Laboratory Medicine, AnSteel Group General Hospital, 2Cancer Science Institute of Singapore, National University of Singapore, 3Department of Pathology, Yong Loo Lin School of Medicine, National University of Singapore, 4Department of Haematology-Oncology, National University Health System

* These authors contributed equally

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.

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Keywords Multiplexed Fluorescent Immunohistochemistry

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