Immunofluorescent staining and imaging of the pupal and adult Drosophila visual system

This immunofluorescence protocol can be used to assay cell morphology, cell positioning and subcellular localization of proteins in the fly eye at stages of development from early pupation to adult. The protocol includes the following procedures: collecting and developmentally staging Drosophila pupae, dissecting fly eyes at defined stages of development, immunostaining of retina and preparing visual system samples (i.e., retina and optic lobe) for confocal microscopy. It is supplemented with images of key dissection steps, guidelines for troubleshooting and examples of data obtained using these methods. Overall, this protocol takes up to 9 d to complete. The amount of hands-on time required on each day varies, ranging from 30 min to several hours depending on the number of stages and/or genotypes one wishes to study.