Optimized protocols for culturing and sectioning mouse intestinal organoids: enhancing efficiency and structural...

Mouse intestinal organoids are essential 3D models that retain host genetic characteristics and complex architecture, making them invaluable for intestinal biology research. However, traditional cultivation and histological processing often suffer from high operational complexity and sample loss during sectioning. This protocol provides an optimized workflow for the establishment of mouse intestinal organoid cultures and their subsequent preparation for immunohistochemistry and fluorescence staining. By refining the handling steps, the method significantly reduces total operational time while enhancing experimental quality. Furthermore, the protocol addresses common challenges in structural integrity, ensuring that the delicate crypt-villus morphology remains intact during the sectioning process. This comprehensive approach offers a robust framework for high-quality histological analysis, supported by a comparative evaluation with traditional methods to guide researchers in different experimental contexts.•Introduces a direct pellet-OCT embedding strategy that minimizes organoid transfer and sample loss during histological processing.•Enables high-quality frozen sections compatible with H&E, Alcian Blue, immunohistochemistry, and immunofluorescence staining.•Provides a practical alternative to conventional agarose-paraffin workflows while preserving organoid morphology and reducing processing complexity.