Whole-mount staining of mouse colorectal cancer organoids and fibroblast-organoid co-cultures

Imaging organoid culture provides an excellent tool for studying complex diseases such as cancer. However, retaining the morphology of intact organoids for immunolabeling has been challenging. Here, we describe a protocol for immunofluorescence staining in intact colorectal cancer organoids derived from mice. We also describe additional steps for co-culture with mouse fibroblasts to enable the study of interactions with other cellular components of the tissue microenvironment. For complete details on the use and execution of this protocol, please refer to Martinez-Ordoñez et al. (2023).1 [Display omitted] •Steps for appropriate organoid seeding and fixation•Protocol for whole-mount staining of 3D organoids grown in semi-suspension and in matrix•Analysis of epithelial-stromal interactions by staining of 3D co-cultures Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Imaging organoid culture provides an excellent tool for studying complex diseases such as cancer. However, retaining the morphology of intact organoids for immunolabeling has been challenging. Here, we describe a protocol for immunofluorescence staining in intact colorectal cancer organoids derived from mice. We also describe additional steps for co-culture with mouse fibroblasts to enable the study of interactions with other cellular components of the tissue microenvironment.