Spatial biology techniques are reshaping modern biomedical research by allowing visualization of cellular organization and interactions within intact tissues. This is of particular importance in hepatology, as liver diseases such as metabolic dysfunction-associated steatohepatitis (MASH), cholangiopathy, fibrosis, and hepatocellular carcinoma are caused by highly heterogeneous multicellular microenvironments. However, widespread adoption of multiplexed imaging techniques is still limited by high cost, specialized equipment, and complex analytical workflows. In a recent study published in electronic gastroenterologyMarlene S. Kohlhepp, Ph.D., Adrien Guillot, Ph.D., and colleagues described a versatile multiplexed immunofluorescence (mIF) workflow designed to overcome these barriers. The authors established an integrated platform and designed an in vitro system that can analyze liver tissue, relying primarily on conventional fluorescence microscopy and open-source computational tools.
Sequential multiplex immunofluorescence using standard laboratory infrastructure
This platform is based on a series of cycles of antibody staining, imaging, and antibody stripping. After each imaging round, antibodies are chemically eluted using a β-mercaptoethanol/SDS-based stripping protocol, allowing additional markers to be applied sequentially to the same sample.
Using this approach, the researchers demonstrated successful detection of approximately 10 to 15 markers within a single specimen while preserving tissue structure over multiple staining cycles. Importantly, the workflow is optimized for formalin-fixed paraffin-embedded (FFPE) liver sections. FFPE liver sections remain the standard material used in routine pathology laboratories.
In this study, we showed that this method allows for simultaneous visualization of multiple hepatocyte populations, including hepatocytes, cholangiocytes, macrophages, endothelial cells, and hepatic stellate cells, thus enabling detailed spatial characterization of the liver microenvironment.
Extending multiplex imaging to organoids and liver-on-a-chip systems
The main innovation of this study was extending multiplexed imaging beyond tissue sections. Researchers adapted this protocol to intrahepatic cholangiocyte organoids, primary hepatocyte cultures, and an advanced liver-on-a-chip platform.
In organoids, multiplex imaging has made it possible to assess epithelial polarity, proliferation, and cell-cell junction integrity by simultaneously analyzing markers such as CK19, β-catenin, ZO-1, Ki67, and PCNA. In primary cell cultures, this method allowed spatial characterization of hepatocytes, macrophages, endothelial cells and stellate cells cultured in the same chamber.
This platform was also successfully implemented in a biliary niche-on-a-chip system containing multiple hepatocyte populations. This demonstrates the feasibility of applying high-dimensional spatial phenotyping to engineered microphysiological systems, an area of increasing importance for disease modeling and drug testing.
CytoPrixm streamlines multidimensional image processing
To address the heavy computational load imposed by sequential multiplexed imaging, the authors developed CytoPrixm, an open-source image processing software package.
CytoPrixm integrates several important preprocessing features, including image stitching, background correction, channel alignment, and DAPI-based image registration. The software is designed to reduce manual workload and improve reproducibility while minimizing the need for advanced coding expertise.
By combining experimental multiplexed imaging with accessible computational infrastructure, this platform aims to facilitate widespread implementation of spatial phenotyping approaches across laboratories.
Implications for translational hepatology
This study highlights the growing importance of spatially resolved analysis in liver disease research. Multiplexed imaging provides mechanistic insights into immune cell recruitment, ductal responses, fibrogenesis, and tissue remodeling while also supporting validation of findings from single-cell and spatial transcriptome datasets.
Importantly, the workflow described in this study focuses on accessibility and scalability rather than maximum marker throughput. By using commercially available reagents, standard fluorescence microscopy, and open-source software, this platform has the potential to significantly lower the barrier to entry for spatial biology research.
As spatially resolved molecular profiling becomes increasingly integrated into translational medicine, such adaptive and cost-effective imaging platforms have the potential to contribute to the development of next-generation digital pathology, disease stratification strategies, and precision hepatology.
sauce:
Jilin University First Hospital
Reference magazines:
Franco Leonardi, B. others. (2026). An integrated platform for multiplexed immunofluorescence across liver tissues and operational models. e Gastroenterology. DOI: 10.1136/egastro-2026-100379. https://egastroenterology.bmj.com/content/4/2/e100379
