Protocol for extended-plex immunofluorescence staining of FFPE tissues using a sequential bleach-and-stain approach

Multiplex immunofluorescence enables spatial analysis of complex biological systems, but increasing plex levels raise cost and workflow complexity. Here, we present a protocol for extending tyramide signal amplification (TSA)-based multiplex immunofluorescence from 6 to 16 markers using a sequential bleach-and-stain approach. It covers tissue preparation, panel establishment, sequential staining, fluorophore bleaching, image acquisition and computational image processing including stitching, registration, and overlay. The workflow is applicable to formalin-fixed paraffin-embedded (FFPE) human cancer and lymphoid tissues and is adaptable through the selection of appropriate antibodies.