Efficient RNA and RNA-protein co-detection in 3D colonoids by whole-mount staining

Here, we describe a protocol to visualize RNA oligos and proteins independently or together using a combination of fluorescence in situ hybridization (FISH) and immunofluorescence in human colonoids, expanding on previously published research. Whole-mount staining is used to preserve the colonoid structure and fix onto glass slides. We describe procedures for efficient plating, fixation, and preservation of the colonoids. This workflow can be adapted to 3D organoid models from other tissues or organisms. For complete details on the use and execution of this protocol, please refer to In et al. (2020). [Display omitted] •Whole-mount staining of 3D colonoids in suspension detect RNA oligos•Whole-mount staining by fixing colonoids onto lysine-coated slides•RNA-protein co-staining in colonoids using suspension or lysine-coated approach Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here, we describe a protocol to visualize RNA oligos and proteins independently or together using a combination of fluorescence in situ hybridization (FISH) and immunofluorescence in human colonoids, expanding on previously published research. Whole-mount staining is used to preserve the colonoid structure and fix onto glass slides. We describe procedures for efficient plating, fixation, and preservation of the colonoids. This workflow can be adapted to 3D organoid models from other tissues or organisms.