Advancements in 3D spheroid imaging: Optimised cryosectioning and immunostaining techniques

This article presents a modified protocol for embedding and sectioning spheroids and organoids, which are increasingly used in research due to their ability to emulate living tissue. The modifi-cations aim to reduce the distortion and damage of these fragile structures during the embedding and sectioning process. The new method involves using optimized embedding containers, a modi-fied embedding protocol, and optimized temperatures for cryosectioning. A heat-induced antigen retrieval protocol was tested and found to significantly increase immunostaining intensity with-out compromising spheroid integrity. The combined approach allowed for the creation of thinner cryosections, leading to clearer and more detailed images. The results suggest that the modified protocol could be widely adopted to enhance the imaging of spheroids and organoids.center dot Paraformaldehyde fixation of spheroids center dot Antigen retrieval treatment of spheroids center dot Embedding in freezing medium and cryosectioning