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URL: https://dx.doi.org/10.17504/protocols.io.bjiukkew

Authors: Jia Ren Lin, Benjamin Izar, Zoltan Maliga, Yu-An Chen, Giorgio Gaglia, Ziming Du, Clarence Yapp, Shaolin Mei, Sandro Santagata, Peter Sorger

Group: Laboratory of Systems Pharmacology, NCIHTAN

Summary: The architecture of normal and diseased tissues strongly influences the development and progression of disease as well as responsiveness and resistance to therapy. We describe a tissue-based cyclic immunofluorescence (t-CyCIF) method for highly multiplexed immunofluorescence imaging of specimens mounted on glass slides.  t-CyCIF generates up to 60-plex images using an iterative process (a cycle) in which conventional low-plex fluorescence images are repeatedly collected from the same sample and then assembled into a high dimensional representation. t-CyCIF requires no specialized instruments or reagents and is compatible with super-resolution imaging; we demonstrate its application to quantifying signal transduction cascades, tumor antigens and immune markers in diverse tissues and tumors. The simplicity and adaptability of t-CyCIF makes it an effective method for pre-clinical and clinical research and a natural complement to single-cell genomics.Key resources, reagents, and software are listed at the HMS LINCS Center Publication Page http://lincs.hms.harvard.edu/lin-elife-2018/ (RRID:SCR_016370). This page provides links to an OMERO image database from which individual images can be obtained; stitched and registered image panels can be obtained at www.cycif.org (RRID:SCR_016267) and a video illustrating the t-CyCIF method can be found at https://vimeo.com/269885646.This protocol is used in the following manuscripts: Lin J-R, Izar B, Wang S, Yapp C, Mei S, Shah P, Santagata S, Sorger PK. (2018). Highly multiplexed immunofluorescence imaging of human tissues and tumors using t-CyCIF and conventional optical microscopes. eLife. PMID: 29993362 Du Z, Lin JR, Rashid R, Maliga Z, Wang S, Aster J, Izar B, Sorger PK, Santagata S. (2019). Qualifying antibodies for image-based immune profiling and multiplexed tissue imaging. Nature Protocols. PMID: 31534232The original t-CyCIF protocol can be found at dx.doi.org/10.17504/protocols.io.rpxd5pn.

Affiliations: Laboratory of Systems Pharmacology, Harvard Medical School, Boston, MA, Laboratory of Systems Pharmacology, Harvard Medical School, Boston, MA;Ludwig Center for Cancer Research at Harvard, Harvard Medical School, Boston, MA, Laboratory of Systems Pharmacology, Harvard Medical School, Boston, MA;Ludwig Center for Cancer Research at Harvard, Harvard Medical School, Boston, MA, Laboratory of Systems Pharmacology, Harvard Medical School, Boston, MA, Laboratory of Systems Pharmacology, Harvard Medical School, Boston, MA;Ludwig Center for Cancer Research at Harvard, Harvard Medical School, Boston, MA;Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, Laboratory of Systems Pharmacology, Harvard Medical School, Boston, MA;Ludwig Center for Cancer Research at Harvard, Harvard Medical School, Boston, MA, Laboratory of Systems Pharmacology, Harvard Medical School, Boston, MA, Laboratory of Systems Pharmacology, Harvard Medical School, Boston, MA, Laboratory of Systems Pharmacology, Harvard Medical School, Boston, MA;Ludwig Center for Cancer Research at Harvard, Harvard Medical School, Boston, MA;Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, Laboratory of Systems Pharmacology, Harvard Medical School, Boston, MA;Ludwig Center for Cancer Research at Harvard, Harvard Medical School, Boston, MA

External URL: www.cycif.org

Version: 2

Publication Date: 2020

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