Preparation of cryo- and paraffin sections of spheroids for histological and immunofluorescence analysis v2

Traditional two-dimensional cell cultures inadequately model cancer progression, prompting increased use of three-dimensional models such as spheroids that better recapitulate tumour microenvironments found in solid tumours, including features such as hypoxia and acidic pH. The small size of spheroids presents challenges for handling and processing using traditional methods. This protocol describes a detailed procedure for preparing spheroid samples for histological staining and immunofluorescence labelling on both cryosections and paraffin sections for light and fluorescence microscopy analysis. The workflow is demonstrated using spheroids derived from normal (SV-HUC-1) and cancerous (T24) human urothelial cell lines but can be readily adapted to spheroids from other cell types and tissue origins. It enables comprehensive morphological and molecular characterization of two-dimensional sections using haematoxylin-eosin staining and immunofluorescence, revealing features such as necrotic cores and differences in cell-cell connectivity.