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iCLAP: an innovative method for integrable co-detection of low-abundance antigens with high-plex immunostaining
Fan Wu1,2, Shuyuan Zheng1, Yani Chen1
1Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland, USA.
Insights
New integrable Co-detection of Low-Abundant Proteins (iCLAP) technology allows sensitive, high-plex protein detection in formalin-fixed, paraffin-embedded tissues. This method enables spatial proteomic studies of low-abundance proteins in complex biological samples.
Area of Science:
- Biotechnology
- Molecular Biology
- Pathology
Background:
- Multiplexed protein imaging is crucial for spatial analysis of complex tissues.
- Detecting low-abundance proteins in formalin-fixed, paraffin-embedded (FFPE) tissues is challenging with current high-plex methods.
- Many important regulators like transcription factors and senescence markers are difficult to study in situ.
Purpose of the Study:
- To develop a sensitive and highly multiplexed protein detection method for FFPE tissues.
- To enable the study of low-abundance proteins in situ within FFPE specimens.
- To expand the capabilities of spatial proteomic analysis in FFPE tissues.
Main Methods:
- Introduced integrable Co-detection of Low-Abundant Proteins (iCLAP).
- iCLAP combines iterative signal amplification with efficient fluorophore inactivation.
- Enabled repeated staining on the same FFPE tissue section for over 40 markers.
Main Results:
- Demonstrated sensitive and highly multiplexed protein detection in FFPE tissues.
- Successfully profiled more than 40 markers on a single FFPE tissue section.
- Revealed spatially distinct senescence-associated protein patterns in human pancreatic tissues.
Conclusions:
- iCLAP significantly enhances the sensitivity of protein detection in FFPE tissues.
- The method facilitates high-dimensional spatial proteomic studies of complex biological processes.
- iCLAP expands the analytical utility of FFPE specimens for in situ protein analysis.
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.
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