Protocol for antibody optimization and panel design in high-dimensional multiplexed immunofluorescence imaging

The rise of spatial biology technologies is advancing our ability to study cellular interactions within native tissue contexts using antibody-based multiplexed imaging. Here, we present a protocol for designing and optimizing a suitable antibody panel, with a specific focus on the PhenoCycler-Fusion system. We outline steps for antibody selection, optimization, and validation, as well as reporter plate preparation and cycle generation for image acquisition. This protocol incorporates key considerations for robust panel development for spatial profiling of tissues. For complete details on the use and execution of this protocol, please refer to Abraham et al.¹.