3D fluorescence staining and confocal imaging of low amount of intestinal organoids (enteroids): Protocol accessible to all.

The emerging field of 3D organ modeling encounters several imaging issues in particular related to antigen retrieval and sample loss during staining processes. Due to their compact shape, several antibodies fail to penetrate intact organoids or spheroids. Histology of organoids can be approached by paraffin inclusion and sectioning at 5 μm as performed for biopsies. However, to fully understand organoid behavior, including cellular organization, extracellular matrix structure, and their response to treatments, 3D imaging is essential. Here we propose an easy workflow allowing (1) immunostaining with a HIER step, (2) preservation of the intact shape of the organoids, (3) sample immobilization in a focal plane reachable for high resolution/short working distance lenses, and (4) minimizing the risk of loss of precious material.