Abstract
Multiplexed protein imaging enables spatial analysis of complex tissues, but detecting proteins expressed at low levels remains challenging, particularly in widely available formalin-fixed, paraffin-embedded (FFPE) specimens. Many biologically important regulators—including senescence markers, transcription factors, and secreted proteins—are therefore difficult to study in situ using existing high-plex methods. Here we show that integrable Co-detection of Low-Abundant Proteins (iCLAP) enables sensitive and highly multiplexed protein detection within the same FFPE tissue section. iCLAP combines iterative signal amplification with efficient fluorophore inactivation, enabling repeated staining of the same tissue section and seamless integration with established multiplex imaging platforms to achieve profiling of more than 40 markers. Application of iCLAP to human pancreatic tissues revealed spatially distinct senescence-associated protein patterns across tissue compartments. Together, iCLAP expands the analytical capabilities of FFPE tissues, enabling high-sensitivity, high-dimensional spatial proteomic studies of complex biological processes.
Data availability
The processed imaging-derived quantitative data generated in this study, together with representative regions of interest (ROIs) extracted from multiplexed whole-slide images, have been deposited in Zenodo (https://doi.org/10.5281/zenodo.18202750, https://doi.org/10.5281/zenodo.18202615, https://doi.org/10.5281/zenodo.18202404, and https://doi.org/10.5281/zenodo.18202295). These data constitute the minimum dataset required to interpret, verify, and extend the findings of this study. Supplementary Data 1 provides human specimen metadata and figure-to-sample mapping necessary for interpretation of the imaging results. Supplementary Data 2 lists all antibodies used for iCLAP immunolabeling, including targets, clones, suppliers, and dilutions. Source Data supporting the quantitative analyses are provided with this article. Raw multiplexed whole-slide imaging data generated from human FFPE tissue sections are not publicly available due to the large size of the datasets and ethical and privacy restrictions associated with human tissue donors and institutional data governance policies. Access to raw whole-slide images may be considered under restricted access for non-commercial research purposes. Requests for access should be directed to the corresponding author and will be reviewed on a case-by-case basis in accordance with institutional policies. Source data are provided with this paper.
Code availability
Custom scripts used for image analysis in this study were developed by the authors. Code necessary to reproduce the analyses presented in this manuscript, including ROI-based quantification and statistical analysis, has been deposited alongside the associated datasets in Zenodo (https://doi.org/10.5281/zenodo.18203368).
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Acknowledgements
The authors thank Xuan Yuan for her contributions to imaging mass cytometry data acquisition at the Mass Cytometry Facility at Johns Hopkins University. This work was supported by the NIH Senescence Network (SenNet) through grants UG3CA275681 and UH3CA275681(PHW), which provided substantial funding for the development of this study. The authors acknowledge the following additional sources of support: U54AR081774 (DW); U54CA268083 (DW); R01CA300052 (DW), U54AG075932 (BS), P30CA006973 (WJH), S10OD034407 (WJH), and 1U54AG075941 (GAK) all from the National Institutes of Health.
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F.W., P.H.W., and D.W. conceived and designed the study, F.W. and S.Y.Z. developed the iCLAP bleaching method, and conducted the experiments. F.W., Y.N.C., P.J.Y., M.J.K., S.L., G.K., S.P., and G.P. contributed to experimental optimization and data acquisition. R.H.Y., B.F.Y., and K.S.H. assisted with image analysis. Q.F.Z. and R.A.A. contributed to the CODEX data acquisition and interpretation. S.M.S., C.C., and W.J.H. contributed to the IMC data acquisition and interpretation. P.R., K.I., C.A.M., N.M., and G.A.K. contributed to the collection and transport of the additional tissue cohort. B.S., L.W., W.J.H., R.A.A., D.W., and P.H.W. provided critical feedback on the methodology. F.W., P.H.W., and D.W. contributed to manuscript writing and editing. DW and PHW supervised the study, secured funding, and provided overall guidance. All authors reviewed and approved the final manuscript.
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Wu, F., Zheng, S., Chen, Y. et al. iCLAP: an innovative method for integrable co-detection of low-abundance antigens with high-plex immunostaining. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69752-y
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DOI: https://doi.org/10.1038/s41467-026-69752-y
