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

TL;DR

IBEX is a versatile, open method enabling high multiplex imaging (>65 parameters) of diverse tissues, compatible with many antibodies and imaging platforms, supporting detailed cellular and tissue analysis.

Contribution

The paper introduces IBEX, a novel iterative immunolabeling and bleaching technique that allows extensive multiplex imaging in tissues with broad compatibility and ease of adoption.

Findings

Supports over 65 parameters in tissue imaging

Compatible with 250+ antibodies and multiple fluorophores

Provides open-source software for image alignment

Abstract

High content imaging is needed to catalogue the variety of cellular phenotypes and multi-cellular ecosystems present in metazoan tissues. We recently developed Iterative Bleaching Extends multi-pleXity (IBEX), an iterative immunolabeling and chemical bleaching method that enables multiplexed imaging (>65 parameters) in diverse tissues, including human organs relevant for international consortia efforts. IBEX is compatible with over 250 commercially available antibodies, 16 unique fluorophores, and can be easily adopted to different imaging platforms using slides and non-proprietary imaging chambers. The overall protocol consists of iterative cycles of antibody labelling, imaging, and chemical bleaching that can be completed at relatively low cost in 2-5 days by biologists with basic laboratory skills. To support widespread adoption, we provide extensive details on tissue processing, curated lists of validated antibodies, and tissue-specific panels for multiplex imaging. Furthermore, instructions are included on how to automate the method using competitively priced instruments and reagents. Finally, we present a software solution for image alignment that can be executed by individuals without programming experience using open source software and freeware. In summary, IBEX is an open and versatile method that can be readily implemented by academic laboratories and scaled to achieve high content mapping of diverse tissues in support of a Human Reference Atlas or other such applications.