Qualifying antibodies for image-based immune profiling and multiplexed tissue imaging

• Published in: Nature protocols Vol. 14; no. 10; pp. 2900 - 2930
• Main Authors: Du, Ziming, Lin, Jia-Ren, Rashid, Rumana, Maliga, Zoltan, Wang, Shu, Aster, Jon C., Izar, Benjamin, Sorger, Peter K., Santagata, Sandro
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.10.2019; Nature Publishing Group
• Subjects: 631/1647/245/2225; 631/250/580; 631/553/2706; 631/67/327; 692/53; Analytical Chemistry; Animals; Antibodies; Antigens; Biological Techniques; Biomarkers, Tumor - immunology; Biomedical and Life Sciences; Computational Biology/Bioinformatics; Data analysis; Enumeration; Fluorescence; Fluorescent antibody technique; Fluorescent Antibody Technique - instrumentation; Fluorescent Antibody Technique - methods; Formaldehyde; Health aspects; Humans; Image Processing, Computer-Assisted - methods; Imaging; Immune checkpoint; Immune system; Immunofluorescence; Immunoglobulins; Immunohistochemistry - methods; Immunological research; Life Sciences; Lymphocytes; Lymphocytes B; Lymphocytes T; Macrophages; Mapping; Measurement; Mice; Microarrays; Multiplexing; Neoplasms - immunology; Neoplasms - pathology; Organic Chemistry; Paraffin; Paraffin Embedding; Paraffins; Protocol; Regulators; Selectivity; Test procedures; Viral antibodies; United States
• ISSN: 1754-2189; 1750-2799; 1750-2799
• DOI: 10.1038/s41596-019-0206-y

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. This protocol provides guidelines for designing and validating antibody panels for fluorescence-based imaging of FFPE tissue sections using cyclic immunofluorescence (t-CyCIF) or other multiplexed imaging methods.