Fly brains were dissected in 1 × PBS solution and fixed in 2% paraformaldehyde S2 media overnight at 4°. Brains were continuously rocked on a nutator throughout the remainder of the protocol. Brains were washed four times for 15 min with PBS with 0.5% Triton X-100 (PBT), blocked at room temperature for 1 hr in PBT with 5% goat serum (PBT-GS). Primary antibodies (1:400 or 1:20) in PBT-GS were incubated overnight at 4°. Brains were then washed four times for 15 min with PBT, and secondary antibodies (1:500) in PBT-GS were incubated at room temperature for 2 hr. Again, brains were washed four times for 15 min with PBT, then mounted in DAPI Fluoromount-G (SouthernBiotech) or ProLong Diamond Antifade Mountant (Invitrogen, Carlsbad, CA).
