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Bex-Plex Enables Simultaneous Visualization of Protein Markers and Ribonucleic Acid Transcripts for Deep Spatial
Samantha Kimmel1, Catherine Murphy1, Patrick Helcl1
1Department of Pediatrics, Division of Allergy, Immunology, and Rheumatology, University of California San Diego.
Journal of Visualized Experiments : Jove
|July 20, 2026
Summary
We developed "Bex-Plex," a new protocol for simultaneous spatial profiling of protein and RNA markers in single cells within intact tissues. This method aids in understanding tumor microenvironments and predicting therapeutic responses.
Area of Science:
- Biotechnology
- Molecular Biology
- Cancer Research
Background:
- Predicting patient response to cancer therapies is challenging due to complex tumor microenvironments.
- Understanding cellular phenotypes, interactions, and spatial organization is crucial for effective treatment design.
- Current methods lack cost-effective ways to profile both transcriptome and proteome with spatial information.
Purpose of the Study:
- To present a novel protocol for simultaneous spatial profiling of protein and RNA markers at single-cell resolution in intact tissues.
- To enable integrated spatial analysis of transcriptional and functional cellular states within the tumor microenvironment.
- To provide a framework for studying spatial biology and predicting therapeutic responsiveness.
Main Methods:
- Combined the iterative bleaching extends multiplexity (IBEX) platform with HiPlex RNAscope technology.
- Developed a modified RNA target retrieval step to preserve nuclear morphology and ensure accurate image alignment.
- Optimized the protocol for fixed frozen tissues, enabling simultaneous visualization of high-plex protein and RNA markers.
Main Results:
- Successfully achieved simultaneous spatial profiling of protein and RNA markers at single-cell resolution.
- Enabled accurate image alignment across cycles, preserving spatial localization information.
- Demonstrated the capability for integrated spatial analysis of transcriptional and functional cellular states.
Conclusions:
- The developed "Bex-Plex" protocol offers a straightforward and cost-effective solution for deep spatial profiling of tumor microenvironments.
- This method provides critical insights into cellular interactions driving therapeutic responsiveness.
- Facilitates advanced research in spatial biology for cancer and other complex tissue systems.
