Mapping Hepatic Stellate Cell Morphology in Mouse Models of Liver Fibrosis.

Tissue-resident fibroblasts are the cellular drivers of fibrosis across tissues. Within the liver, hepatic stellate cells (HSCs), the resident pericytes with their unique "star-like" morphology, activate into fibrogenic myofibroblasts during injury to produce the hepatic scar. While decades of work elucidated major signaling pathways underlying HSC activation, the functional significance of their distinct dendritic morphology remains elusive due to technical limitations in imaging and analysis. This protocol presents a comprehensive imaging pipeline to visualize and model HSC morphology in intact liver tissue. This approach combines fluorescent labeling of HSCs using mouse genetics, in situ liver perfusion-based tissue preparation, and a modified iDISCO tissue clearing method to achieve optical transparency in tissue specimens that can be several centimeters thick. Cleared samples are imaged using optimized confocal microscopy platforms, and image datasets are processed through a customized analysis workflow for three-dimensional reconstruction of individual HSCs at single-cell resolution. Together, this pipeline enables reproducible mapping of HSC morphology within the complex liver microenvironment, including their dendritic processes and spatial relationships to neighboring cells. Beyond the liver, the workflow is adaptable to fibroblasts in other organs, such as the lung, kidney, intestine, and heart, where resident mesenchymal cells exhibit similar fibrogenic roles in disease. The proposed framework, therefore, establishes a methodological foundation for comparative cross-organ studies to probe fibroblast structure-function relationships and therapeutic vulnerabilities in fibrotic disease.