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High content imaging is needed to catalogue the variety of cellularphenotypes and multi-cellular ecosystems present in metazoan tissues. Werecently developed Iterative Bleaching Extends multi-pleXity (IBEX), aniterative immunolabeling and chemical bleaching method that enables multiplexedimaging (>65 parameters) in diverse tissues, including human organs relevantfor international consortia efforts. IBEX is compatible with over 250commercially available antibodies, 16 unique fluorophores, and can be easilyadopted to different imaging platforms using slides and non-proprietary imagingchambers. The overall protocol consists of iterative cycles of antibodylabelling, imaging, and chemical bleaching that can be completed at relativelylow cost in 2-5 days by biologists with basic laboratory skills. To supportwidespread adoption, we provide extensive details on tissue processing, curatedlists of validated antibodies, and tissue-specific panels for multipleximaging. Furthermore, instructions are included on how to automate the methodusing competitively priced instruments and reagents. Finally, we present asoftware solution for image alignment that can be executed by individualswithout programming experience using open source software and freeware. Insummary, IBEX is an open and versatile method that can be readily implementedby academic laboratories and scaled to achieve high content mapping of diversetissues in support of a Human Reference Atlas or other such applications.

IBEX: An open and extensible method for high content multiplex imaging of diverse tissues