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

Claire Charlet-Faure; Annemette Præstegaard Thulesen; Adelina Rogowska-Wrzesinska

doi:10.1016/j.mex.2023.102415

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

MethodsX. 2023 Oct 6:11:102415. doi: 10.1016/j.mex.2023.102415. eCollection 2023 Dec.

Authors

Claire Charlet-Faure^{1

2}, Annemette Præstegaard Thulesen¹, Adelina Rogowska-Wrzesinska¹

Affiliations

¹ Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, Odense M DK-5230, Denmark.
² Faculté des Sciences Site St Charles Aix Marseille Université, 3 place Victor Hugo, Marseille 13331, France.

Abstract

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 modifications 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 modified embedding protocol, and optimized temperatures for cryosectioning. A heat-induced antigen retrieval protocol was tested and found to significantly increase immunostaining intensity without 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.•Paraformaldehyde fixation of spheroids•Antigen retrieval treatment of spheroids•Embedding in freezing medium and cryosectioning.

Keywords: Antigen retrieval; Antigen retrieval and cryosectioning of multicellular 3D spheroids; Cryosectioning; Fluorescent microscopy; Immunostaining; Organoids; Spheroids.