After the recordings, organoid slices were transferred into a fixative solution containing 4% paraformaldehyde and 0.2% picric acid in 0.1M phosphate buffer for 24 hours, and subsequently stained with Alexa Fluor 594 Streptavidin (Jackson ImmunoResearch; 1:500) to detect biocytin. Slices were mounted in Vectashield Medium (Vector Laboratories) and subjected to Z-stack image acquisitions using Nikon C1 confocal microscope (objective: 40×/1.3, Plan Fluor).
