A Simplified and Robust Immunofluorescence Labeling Method for Complex 3D Cell Cultures: Minimizing Manipulation and Maximizing Data in Whole-Mount Analysis of Organoids, Spheroids, and Co-culture Models.

Whole-mount imaging and labeling of 3D organoids and spheroids offer unparalleled insights into spatial biology, organogenesis, disease mechanisms, and cancer cell dynamics within a physiologically relevant 3D context. Those techniques in biomedical research, yet its application to complex 3D cell culture models, remain fraught with challenges. Current methods often suffer from sample damage and loss, in addition to lengthy and complex protocols involving multiple steps and reagent preparations. To address these challenges, we describe a novel approach that overcomes limitations of conventional techniques by preserving sample integrity, minimizing sample manipulation, and eliminating the need for multiple reagents. The current method facilitates comprehensive sample analysis, significantly improving the efficiency and accuracy of protein visualization. It is compatible with a wide range of samples, including organoids and spheroids in hydrogels, organ-on-chip models, and co-culture systems.