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Imaging Cell Signaling in Tissues Using the IBEX Method
Yuxuan Meng1, Changtai Li1, Gregory S Patient1
1UCL Institute of Ophthalmology, London, UK.
Insights
Highly multiplexed immunohistochemistry, like the innovative IBEX method, improves cell signaling studies by mapping protein locations in tissues. This protocol simplifies spatial biology research for better understanding tissue interactions.
Area of Science:
- Tissue biology and spatial omics
- Cellular signaling and molecular interactions
Background:
- Understanding cell signaling in tissues requires precise localization of multiple proteins simultaneously.
- Existing methods may lack the multiplexing capacity or simplicity for comprehensive spatial analysis.
- Integrating protein data with tissue context (stroma, adjacent structures) is crucial for biological insights.
Purpose of the Study:
- To present a simplified protocol for the In-situ Biodistribution by Exchange (IBEX) method for highly multiplexed immunohistochemistry on tissues.
- To demonstrate the utility of IBEX for simultaneously localizing numerous cell signaling pathways within a tissue microenvironment.
- To provide guidance for applying IBEX, showcasing its potential for spatial biology research.
Main Methods:
- Adaptation and simplification of the IBEX (In-situ Biodistribution by Exchange) cyclic immunohistochemistry protocol for tissue analysis.
- Application of a 12-marker antibody panel for simultaneous detection of multiple proteins.
- Utilizing human retina tissue as a model system to validate the protocol's effectiveness and demonstrate spatial protein distribution.
Main Results:
- Successful implementation of a simplified IBEX protocol for highly multiplexed immunohistochemistry on tissue samples.
- Demonstrated simultaneous localization of 12 different protein markers within the human retina.
- Provided a clear visualization of protein spatial distribution and cellular context within the tissue.
Conclusions:
- The simplified IBEX method offers an accessible and powerful approach for high-plex spatial profiling in tissues.
- This technique enhances the study of cell signaling by revealing the spatial organization of molecular pathways.
- IBEX facilitates a deeper understanding of tissue architecture, cell interactions, and disease mechanisms through spatial omics.
Abstract:
The study of cell signaling within tissues can be enhanced using highly multiplexed immunohistochemistry to localize the presence and spatial distribution of numerous pathways of interest simultaneously. Additional data can also be gained by placing the identified proteins into the context of adjacent structures, stroma, and interacting partners. Here, we outline a protocol for using the recently described IBEX method on tissues. This is an open and simple cyclic immunohistochemistry approach suited to this application. We describe a simplified protocol and provide guidance on the method, using a 12-marker panel on human retina to demonstrate the approach.
