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https://doi.org/10.3791/58711
Title: | Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma | Authors: | Hong, Guo Fan, Shuangyi Phyu, The Maheshwari, Priyanka Hoppe, Michal Marek Hoang, Mai Phuong de Mel, Sanjay Poon, Michelle Ng, Siok-Bian Jeyasekharan, Anand D |
Keywords: | Science & Technology Multidisciplinary Sciences Science & Technology - Other Topics Immunology and Infection Issue 143 Multiplexed immunofluorescence multispectral imaging quantitative analysis biomarkers lymphoma digital pathology TYRAMIDE SIGNAL AMPLIFICATION |
Issue Date: | 1-Jan-2019 | Publisher: | JOURNAL OF VISUALIZED EXPERIMENTS | Citation: | Hong, Guo, Fan, Shuangyi, Phyu, The, Maheshwari, Priyanka, Hoppe, Michal Marek, Hoang, Mai Phuong, de Mel, Sanjay, Poon, Michelle, Ng, Siok-Bian, Jeyasekharan, Anand D (2019-01-01). Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma. JOVE-JOURNAL OF VISUALIZED EXPERIMENTS 2019 (143). ScholarBank@NUS Repository. https://doi.org/10.3791/58711 | 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. | Source Title: | JOVE-JOURNAL OF VISUALIZED EXPERIMENTS | URI: | https://scholarbank.nus.edu.sg/handle/10635/207278 | ISSN: | 1940087X | DOI: | 10.3791/58711 |
Appears in Collections: | Staff Publications Elements |
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Hong G. Jove 2019. MIF analysis in lymphoma.pdf | Published version | 3.44 MB | Adobe PDF | CLOSED | Published |
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