A new study published in eGastroenterology on June 26 describes a multiplex immunofluorescence workflow designed to advance spatial research in liver biology. Dr. Marlene S. Kohlhepp, Dr. Adrien Guillot, and colleagues developed an integrated platform that enables the analysis of liver tissues and engineered in vitro systems using conventional fluorescence microscopy and open-source computational tools.
The platform employs sequential cycles of antibody staining, imaging, and chemical stripping with a β-mercaptoethanol/SDS-based protocol. This allows researchers to apply multiple markers—approximately 10–15 per specimen—while preserving tissue architecture through several staining cycles. The workflow is optimized for formalin-fixed paraffin-embedded (FFPE) liver sections commonly used in pathology laboratories.
According to the authors, this method enables simultaneous visualization of various hepatic cell populations such as hepatocytes, cholangiocytes, macrophages, endothelial cells, and hepatic stellate cells within a single sample. The approach was also adapted for use with intrahepatic cholangiocyte organoids, primary liver cell cultures, and advanced liver-on-a-chip platforms.
In organoid models, multiplex imaging facilitated assessment of epithelial polarity and proliferation by analyzing markers like CK19, β-catenin, ZO-1, Ki67, and PCNA together. In primary cell cultures grown within one chamber or biliary niche-on-a-chip systems containing multiple cell types, the technique allowed detailed spatial characterization.
To address data processing challenges from sequential imaging rounds, the team developed CytoPrixm—an open-source software package integrating image stitching, background correction, channel alignment, and DAPI-based registration functions to streamline workflows without requiring advanced coding skills.
The study concludes that coupling experimental multiplex imaging with accessible computational infrastructure could broaden adoption of spatial phenotyping methods across laboratories. By relying on commercially available reagents and standard microscopes alongside open-source software rather than specialized equipment or maximal marker throughput strategies alone, the workflow aims to lower barriers for researchers investigating complex multicellular environments relevant to disease modeling and translational medicine.
