Lin, Jia-RenRashid, RumanaMaliga, ZoltanIzar, BenjaminSantagata, SandroDu, ZimingWang, ShuAster, JonSonger, Peter2019-12-182019-09-18Du, 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: 2900-930.1754-21891750-2799http://nrs.harvard.edu/urn-3:HUL.InstRepos:42068905Multiplexed 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.en-USGeneral Biochemistry, Genetics and Molecular BiologyJournal Article2019-12-1810.1038/s41596-019-0206-y