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Spatial mapping of protein composition and tissue organization: a primer for multiplexed antibody-based imaging


Tissues and organs are composed of distinct cell types that must operate in concert to perform physiological functions. Efforts to create high-dimensional biomarker catalogs of these cells have been largely based on single-cell sequencing approaches, which lack the spatial context required to understand critical cellular communication and correlated structural organization. To probe in situ biology with sufficient depth, several multiplexed protein imaging methods have been recently developed. Though these technologies differ in strategy and mode of immunolabeling and detection tags, they commonly utilize antibodies directed against protein biomarkers to provide detailed spatial and functional maps of complex tissues. As these promising antibody-based multiplexing approaches become more widely adopted, new frameworks and considerations are critical for training future users, generating molecular tools, validating antibody panels, and harmonizing datasets. In this Perspective, we provide essential resources, key considerations for obtaining robust and reproducible imaging data, and specialized knowledge from domain experts and technology developers.

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Fig. 1: Obtaining high-content imaging data using a wide range of multiplexed antibody-based imaging platforms.
Fig. 2: Considerations for the choice and implementation of multiplexed antibody-based imaging technologies into existing workflows.
Fig. 3: Phases of panel development and validation for multiplexed antibody-based imaging assays.
Fig. 4: Process of conjugating antibodies with modifiers for multiplexing.


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We are grateful for engaging and thoughtful discussions from the Affinity Reagent Imaging and Validation Working Group, HuBMAP Consortium. The authors would like to acknowledge funding from the following sources: NIH U54 DK120058, NIH U54 EY032442, NIH R01 AI145992, NIH R01 AI138581 (R. M. C. and J. M. S.), NIH T32ES007028 (E. K. N), NIH U54 HG010426-01 (M. P. S. and G. P. N.), NIH UG3 HL145600-01, NIH UH3 CA246633-01 (R. M. A), NIH UH3 CA246635-01 (N. L. K.), Swedish Research Council 2018-06461 (E. L.), Erling Persson Family Foundation (E. L.), Wallenberg Foundation (E. L.), NIH UH3 CA246594-01 (A. S. and E. M.), NIH T32CA196585 and ACS PF-20-032-01-CSM (J. W. H.), and NIH UH3 CA255133-03 (S.K.S.) and European Molecular Biology Laboratory (S. K. S.). This work was supported, in part, by the Intramural Research Program of the NIH, NIAID and NCI. We thank J. Hernandez and J. Davis (National Cancer Institute, NIH) for providing de-identified human tissues featured in this work, and K. Prummel (EMBL) for comments on the manuscript. Figures were made using the tools on

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J. W. H., E. K. N., A. J. R, J. M. C., and S. K. S. conceived the plan, designed figures, and wrote the manuscript. R. T. B. designed display items and helped write the manuscript. A. A., K. C., E. M., J. H., A. E. W., J. F., J. C., A. S., R. M. C., R. M. A., G. P. N., K. C., S. M. H., R. N. G., J. M. S., E. L., M. P. S., and N. L. K. provided domain expertise and assisted with the conception and writing of the manuscript.

Corresponding authors

Correspondence to Andrea J. Radtke or Sinem K. Saka.

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Competing interests

A. E. W. is an employee and shareholder of Abcam plc. J. F. is an employee of Cell Signaling Technologies. J. C. is an employee and shareholder of BioLegend. J. H. is an employee of Bio-Techne. E. S. is an employee of Thermo Scientific. E. M. and A. S. are current or past employees of GE Research. K. C. is an inventor for patent applications covering some technologies described in this paper and a cofounder of LifeCanvas Technologies. G. P. N. is inventor on a US patent, covering some technologies described in this paper, has equity in and/or is a member of the scientific advisory board of Akoya Biosciences. S. K. S. is an inventor for patent applications related to some of the methods described here.

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Peer review information Nature Methods thanks Trevor McKee, David Rimm and Takahiro Tsujikawa for their contribution to the peer review of this work. Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Supplementary information

Supplementary Information

Supplementary Fig. 1, Tables 1–6 and References

Reporting Summary

Supplementary Dataset

List of community-validated antibody clones targeting common human and mouse protein markers across different highly multiplexed imaging platforms

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Hickey, J.W., Neumann, E.K., Radtke, A.J. et al. Spatial mapping of protein composition and tissue organization: a primer for multiplexed antibody-based imaging. Nat Methods (2021).

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