Unified platform for multiplex immunofluorescence across liver tissues and engineered models.

Area of Science:

• Hepatology
• Immunohistochemistry
• Digital Pathology

Background:

• Multiplex immunostaining and digital image analysis are crucial for studying liver disease microenvironments.
• Technological complexity, specialized equipment, and incompatibility with standard workflows hinder widespread adoption of multiplexing platforms.
• Existing methods often do not work with archival formalin-fixed paraffin-embedded (FFPE) tissues or in vitro cell cultures.

Purpose of the Study:

• To develop a user-friendly multiplex immunofluorescence workflow adaptable to various sample types.
• To enable detailed spatial mapping of cellular markers in liver research.
• To overcome barriers preventing the integration of multiplexing into routine laboratory practices.

Main Methods:

• A customized immunofluorescence workflow using sequential antibody stripping was created for FFPE liver sections, organoids, and primary mouse liver cells.
• Cells were cultured in 2D systems or a biliary niche-on-a-chip platform.
• An open-source, Python-based software tool was used for image processing and channel alignment.

Main Results:

• The protocol successfully generated multiplexed digital images from diverse FFPE liver samples and cell cultures.
• The workflow visualizes antigen-expressing cells and protein-protein interactions via proximity ligation assays.
• Technical considerations for integrating this method into standard immunohistochemistry workflows are detailed.

Conclusions:

• This methodology integrates multiplex immunofluorescence into standard laboratory workflows.
• It enables researchers to overcome challenges related to technological complexity and cost.
• Facilitates the transition from conventional to multiplexed microscopy for broader accessibility.