Qualifying Antibodies for Image-Based Immune Profiling and Multiplexed Tissue Imaging
Author
Du, Ziming
Wang, Shu
Songer, Peter
Published Version
https://doi.org/10.1038/s41596-019-0206-yMetadata
Show full item recordCitation
Du, Ziming, Jia-Ren Lin, Rumana Rashid, Zoltan Maliga, Shu Wang, Jon C. Aster, Benjamin Izar, Peter K. Sorger, and Sandro Santagata. 2019. Qualifying Antibodies for Image-based Immune Profiling and Multiplexed Tissue Imaging. Nature Protocols 14, no. 10: 2900-930.Abstract
Multiplexed tissue imaging enables precise, spatially resolved enumeration and characterization of cell types and states in human resection specimens. A growing number of methods applicable to formalin-fixed, paraffin-embedded (FFPE) tissue sections have been described, the majority of which rely on antibodies for antigen detection and mapping. This protocol provides step-by-step procedures for confirming the selectivity and specificity of antibodies used in fluorescence-based tissue imaging and for the construction and validation of antibody panels. Although the protocol is implemented using tissue-based cyclic immunofluorescence (t-CyCIF) as an imaging platform, these antibody-testing methods are broadly applicable. We demonstrate assembly of a 16-antibody panel for enumerating and localizing T cells and B cells, macrophages, and cells expressing immune checkpoint regulators. The protocol is accessible to individuals with experience in microscopy and immunofluorescence; some experience in computation is required for data analysis. A typical 30-antibody dataset for 20 FFPE slides can be generated within 2 weeks.Terms of Use
This article is made available under the terms and conditions applicable to Other Posted Material, as set forth at http://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAACitable link to this page
http://nrs.harvard.edu/urn-3:HUL.InstRepos:42068905
Collections
- HMS Scholarly Articles [17922]
Contact administrator regarding this item (to report mistakes or request changes)