Iterative Bleaching Extends Multiplicity with Use of Staining Automation for Core Facilities

Iterative Bleaching Extends Multiplicity (IBEX) is a highly sensitive tyramide signal amplification (TSA)-based multi-round fluorescence imaging technique enabling detailed examination of numerous protein biomolecules in a single tissue sample through repeated bleaching, re-staining, and image co-registration. Despite its growing adoption for generating highly multiplexed immunostaining, implementing IBEX in an automated fashion through deployment in core facilities offers numerous benefits, including increased throughput, reproducibility, and overall efficiency of laboratory staff. Here, we demonstrate the successful application of IBEX on two broadly utilized autostainer platforms and discuss important optimization considerations for panel design using TSA-based reactions for formalin-fixed, paraffin-embedded (FFPE) tissue samples. This includes the methodology for testing antibody-antigen retrieval sensitivity, an important consideration for IBEX panel design when determining the sequence of antibody development. Additionally, we showcase downstream imaging approaches utilizing commercially available whole-slide scanners and illustrate quality alignment and analysis using commercially available cell phenotyping software. Through detailed descriptions of these methodologies and accompanying videos, we aim to highlight the versatility and ease of IBEX, offering practical guidance for core pathology facilities to integrate IBEX into their existing automated staining platforms.