Dissection of Drosophila brains

The brains of 3- to 5-d old female Drosophila flies were removed using a standard dissecting technique (Wu and Luo, 2006) on a Sylgard (Dow Corning) dish after brief anesthesia on a CO₂ pad. Females were chosen to keep sexual dimorphisms consistent between experiments. Using Dumont #5 forceps (Fine Science Tools, 11251-10), heads were removed from the body and placed in HL3.1 solution. The proboscis was then removed to gain access to the inside of the cuticle. Carefully tearing away at the cuticle until the brain is released, the brains were cleared of all tracheal tissue. Dissected brains were then mounted in ∼10 μl of HL3.1 on a glass slide (Superfrost, ThermoFisher), and sealed shut using a 25-mm square cover glass (Menzel–Gläser, ThermoFisher) rimmed with silicone vacuum grease (Dow Corning) with a paintbrush. For fixed brain imaging, brains were dissected as usual and then fixed in 4% paraformaldehyde (PFA) for 40 min and then washed in HL3.1 solution. Brains were then mounted in the same manner and imaged.