Sp5 2 sted cw confocal microscope

The protocol was adapted from the Flylight protocol [28] . Briefly, brains from 1-to 2-day old flies were dissected in cold S2 media and fixed with 2% paraformaldehyde for 55 minutes. After a brief rinse with 0.5% Triton X-100, brains were blocked with blocking solution (5% normal donkey serum and 0.5% TritonX-100 in phosphate-buffered saline) for 1.5 hours and then were incubated with primary antibodies (Supplementary Table 2). Signals for hPNPO were amplified with the Tyramide SuperBoost kit (Invitrogen, Cat # B40926) by following the manufacturer's protocol. After incubation with the secondary antibodies and washes, brains were mounted on a double-frosted slide and covered with coverslips. DAPI was added into the wash buffer to stain the nucleus when needed. Images were taken using Leica SP5-II-STED-CW confocal microscope in the Integrated Light Microscopy Core Facility at the University of Chicago and processed in Fiji [29] .