Immunofluorescent staining of Drosophila larval brain tissue

The Drosophila larval brain is a well-established model for investigating the role of stem cells in development. Neuroblasts (neural stem cells) must be competent to generate many thousands of differentiated neurons through asymmetric divisions during normal development. Studies in fly neuroblasts have been instrumental in identifying how the establishment and maintenance of cell polarity influence cell fate, and they have produced a wide array of molecular cell-polarity markers. Moreover, neuroblasts and their progeny can be positively identified using a variety of cell-fate markers. This article describes procedures for the collection and processing of Drosophila larval brains for examination by immunolocalization of cell-fate and cell-polarity markers. The protocol can be used for dissecting, fixing, and staining brains from larvae at any developmental stage. The number of brains processed using this method is limited only by how many brains can be dissected in 20 min, which is the maximum amount of time dissected tissues should remain in buffer before fixation. This protocol can be used for simultaneous costaining of multiple proteins.