Histological Processing of Organoids for Immunostaining.

Organoids are three-dimensional cell structures derived from stem cells that recapitulate the architecture and function of native tissues. Histological analysis of organoids is essential for assessing their structure, cellular composition, and responses to experimental conditions. However, their small size and fragility make standard paraffin embedding workflows difficult. Here, we describe a robust and reproducible protocol for the fixation, paraffin embedding, and sectioning of human organoids, enabling high-quality histological and immunostaining analysis. The method involves direct fixation within the culture matrix and inclusion in HistoGel to prevent organoid loss during processing. The protocol is compatible with hematoxylin-eosin (H&E) staining and multiplex immunofluorescence. Critical steps, troubleshooting, and adaptations for intestinal and cardiac organoids are discussed. This cost-effective and accessible method supports long-term preservation and detailed structural analysis of organoid models. Key features • Enables reliable fixation, embedding, and sectioning of fragile human organoids for histological and immunofluorescence analysis. • Uses HistoGel inclusion to prevent organoid loss during paraffin embedding and processing. • Compatible with multiple organoid types (intestinal, cardiac, etc.) and staining methods (H&E, immunofluorescence). • Produces high-quality paraffin blocks and sections suitable for long-term storage.