Wholemount Immunofluorescence of Organoids (ver 3.0) v1

This protocol is for wholemount immunofluorescence of organoids, co-cultures or other 3-dimensional structures that can be grown in scaffolds. Stained organoids are then cleared and imaged with a laser-scanning confocal or lightsheet microscope depending on equipment availability and needs. Optionally, samples can also be cheaply counterstained for amines using the FLARE Protocol (Lee et al., Nature Protocols 2022) before immunostaining is performed. S-phase entry can also be visualised by EdU dosing during the cell culture, and later staining with click detection kits of your choice (the Click-iT EdU are incredibly bright and I prefer them to Ki67 immuno). Cleared samples can be washed after imaging and processed to paraffin if histological characterisation is required.