# iCLAP: an innovative method for integrable co-detection of low-abundance antigens with high-plex immunostaining

## TL;DR

iCLAP is a new method that improves detection of low-abundance proteins in preserved tissues, allowing detailed spatial analysis of complex biological processes.

## Contribution

The novel iCLAP method enables sensitive, high-plex detection of low-abundance proteins in FFPE tissues through iterative staining and fluorophore inactivation.

## Key findings

- iCLAP allows detection of over 40 protein markers in a single FFPE tissue section.
- The method reveals spatially distinct senescence-associated protein patterns in human pancreatic tissues.
- iCLAP integrates with existing high-plex imaging platforms for enhanced profiling.

## 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.

Wu and colleagues present an integrated imaging workflow that combines signal amplification with multiplex immunostaining to improve detection of low-abundance proteins in human tissues, enabling detailed spatial profiling across many molecular markers.