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IBEX: an iterative immunolabeling and chemical bleaching method for high-content imaging of diverse tissues

Nature Protocols volume 17, pages 378–401 (2022)Cite this article

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Abstract

High-content imaging is needed to catalog the variety of cellular phenotypes and multicellular ecosystems present in metazoan tissues. We recently developed iterative bleaching extends multiplexity (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 >250 commercially available antibodies and 16 unique fluorophores, and can be easily adopted to different imaging platforms using slides and nonproprietary imaging chambers. The overall protocol consists of iterative cycles of antibody labeling, imaging and chemical bleaching that can be completed at relatively low cost in 2–5 d 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 a noncommercial 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.

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Fig. 1: Overview of tissue grossing, sample preparation, IBEX protocols and image registration workflow.
Fig. 2: Schematic overview of automated IBEX protocol.
Fig. 3: Representative images of manual IBEX method in human tissues.
Fig. 4: Representative images of automated IBEX method in human tissues.

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Data availability

The datasets generated during the current study are available in the Zenodo repository (https://doi.org/10.5281/zenodo.5244551).

Code availability

All custom code used in this work is freely available as open-source software under the Apache 2.0 license from the National Institute of Allergy and Infectious Diseases (NIAID) GitHub organization. The registration algorithm is available from https://github.com/niaid/sitk-ibex, and the Imaris extension code is available from https://github.com/niaid/imaris_extensions.

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Acknowledgements

This research was supported by the Intramural Research Program of the National Institutes of Health (NIH), NIAID and National Cancer Institute (NCI). This research was also partially supported by a Research Collaboration Agreement (RCA) between NIAID and BioLegend, Inc. (RCA# 2020-0333) and the Chan Zuckerberg Initiative Human Cell Atlas Thymus Seed Network. C.J.C is supported as a UK-US Fulbright Scholar and Fight for Sight Research Scholar. Z.Y. and B.C.L. are supported by the Bioinformatics and Computational Biosciences Branch (BCBB) Support Services Contract HHSN316201300006W/HHSN27200002 to Medical Science & Computing, LLC. D.J. is supported by the grant of the European Research Council (ERC); European Consolidator Grant, XHale (reference #771883). We would like to thank R. Pelletier and M. Aruda from Fluigent for their sterling assistance with the ARIA fluidics device. We are grateful for the technical support provided by G. Portugal, E. Cox and E. Buck from Harvard Apparatus. We thank Dr. S. Pittaluga for her assistance with tissue grossing and orientation. We are appreciative of Drs. G. Cattoretti and M. Bolognesi for sharing their insights on fluorophore inactivation with sodium borohydride.

Author information

Author notes

  1. These authors contributed equally: Andrea J. Radtke, Colin J. Chu.

Authors and Affiliations

  1. Center for Advanced Tissue Imaging, Laboratory of Immune System Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

    Andrea J. Radtke, Rebecca T. Beuschel & Ronald N. Germain

  2. Lymphocyte Biology Section, Laboratory of Immune System Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

    Colin J. Chu, Hiroshi Ichise, Anita Gola, Emily Speranza, Nishant Thakur & Ronald N. Germain

  3. Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, UK

    Colin J. Chu

  4. Bioinformatics and Computational Bioscience Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

    Ziv Yaniv & Bradley Lowekamp

  5. Howard Hughes Medical Institute, Chevy Chase, MD, USA

    Li Yao

  6. Leica Microsystems Inc., Wetzlar, Germany

    James Marr

  7. Howard Hughes Medical Institute, Robin Neustein Laboratory of Mammalian Cell Biology and Development, The Rockefeller University, New York, NY, USA

    Anita Gola

  8. Biological Imaging Section, Research Technologies Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

    Juraj Kabat

  9. Innate Immunity and Pathogenesis Section, Laboratory of Virology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, USA

    Emily Speranza

  10. Department of Business Development, BioLegend, Inc, San Diego, CA, USA

    Joshua Croteau

  11. Institute of Pathology, Hannover Medical School, Member of the German Center for Lung Research (DZL), Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Hannover, Germany

    Danny Jonigk

  12. Surgical Oncology Program, Metastasis Biology Section, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

    Jeremy L. Davis & Jonathan M. Hernandez

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Contributions

A.J.R, C.J.C. and R.N.G. wrote the manuscript. A.J.R., C.J.C., H.I. and R.T.B. designed and executed the experiments. Z.R.Y. and B.L. developed image analysis software. L.Y. designed Figs. 1 and 2, and A.J.R. designed Figs. 3 and 4. A.G., A.J.R. and J.K. prepared supplementary videos. J.M. integrated the Leica microscope with the fluidics device. E.S., N.T., J.C., D.J., J.L.D. and J.M.H. provided technical insight, reagents and tissues. All authors offered guidance for the development and optimization of the workflows.

Corresponding authors

Correspondence to Andrea J. Radtke or Ronald N. Germain.

Ethics declarations

Competing interests

J.C. is an employee of Biolegend, Inc., and J.M. is an employee of Leica Microsystems, Inc.

Peer review

Peer review information

Nature Protocols thanks Fan Zhang, Yongxin Zhao and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Radtke, A. J. et al. Proc. Natl Acad. Sci. USA 117, 33455–33465 (2020): https://doi.org/10.1073/pnas.2018488117

Gola, A. et al. Nature 589, 131–136 (2021): https://doi.org/10.1038/s41586-020-2977-2

Speranza, E. et al. Preprint at bioRxiv (2021): https://doi.org/10.1101/2021.09.08.459430

Extended data

Extended Data Fig. 1 Critical steps in the manual IBEX protocol.

a, Photo depicting central placement of tissue within a two-well chambered coverglass. Glass surface is coated with chrome gelatin alum (invisible when fully dry). b, Picture of small bubbles that form during successful LiBH4 treatment. c, Visual instructions on how to match unique nuclear shapes (Hoechst in yellow, blue box) across the imaging volumes. The left image corresponds to a live image in the Leica LAS X Navigator software. The right image corresponds to the image captured from the previous IBEX cycle. Red circles indicate that the described alignment procedure is being done at the first z-slice (‘Begin’) of the z-stack.

Extended Data Fig. 2 Equipment and assembly of imaging chamber for automated IBEX protocol.

a, Tissues are sectioned onto coated 22 × 22 mm square coverslips and assembled into the RC-21B Large Closed Bath Imaging Chamber. b,c, Top view of imaging chamber placed into PM-2 Platform for Series 20 chambers without (b) and with (c) magnetic platform clamp. d, Equipment footprint of automated IBEX setup; FLPG. e, Complete assembly of PM-2 Platform with RC-21B chamber onto SA-20PL Series 20 stage adapter. Fluid inlet and vacuum outlet highlight the fluid path. Heating electrodes are attached to the top and bottom of the platform using metal prongs that must be bent by user to allow placement into the stage. Temperature probe is inserted into small hole at top of platform to maintain 37 °C for the duration of the protocol.

Supplementary information

Supplementary Information

Tables 1–4 and legends for Supplementary Videos 1–7.

Reporting Summary

Supplementary Video 1

High dimensional imaging of human lymph node using manual IBEX method. Confocal images of human mesenteric lymph node from a 9 cycle 38 parameter IBEX experiment with Hoechst serving as a fiducial.

Supplementary Video 2

High dimensional imaging of human spleen using manual IBEX method. Confocal images of human spleen from a 4 cycle 25 parameter IBEX experiment with Hoechst serving as a fiducial.

Supplementary Video 3

High dimensional imaging of human liver using manual IBEX method. Confocal images of human liver from a 4 cycle 22 parameter IBEX experiment with Hoechst serving as a fiducial.

Supplementary Video 4

High dimensional imaging of human lymph node using automated IBEX method. THUNDER widefield images of human mesenteric lymph node from a 6 cycle 24 parameter IBEX experiment with Hoechst serving as a fiducial.

Supplementary Video 5

High dimensional imaging of human jejunum using automated IBEX method. THUNDER widefield images of human jejunum from a 6 cycle 24 parameter IBEX experiment with Hoechst serving as a fiducial.

Supplementary Video 6

High dimensional imaging of human skin using automated IBEX method. THUNDER widefield images of human skin from a 5 cycle 19 parameter IBEX experiment with Hoechst serving as a fiducial.

Supplementary Video 7

High dimensional imaging of human kidney (FFPE) using automated IBEX method. THUNDER widefield images of human FFPE kidney sections from a 5 cycle 16 parameter IBEX experiment with Hoechst serving as a fiducial.

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Radtke, A.J., Chu, C.J., Yaniv, Z. et al. IBEX: an iterative immunolabeling and chemical bleaching method for high-content imaging of diverse tissues. Nat Protoc 17, 378–401 (2022). https://doi.org/10.1038/s41596-021-00644-9

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