Protocol for labeling Drosophila brains

Single and double channel labeling of Drosophila brains was carried out as previously described (Kohl et al. 2014). For labeling of UAS-LA::Halo2 fillet preparation of wandering third instar larvae were made followed by the same protocol used for labeling whole brains. For detailed information on staining Chemical Brainbow brains and antennal segments, see Supplemental Materials and Methods in File S1. We find that CLIPf substrates weakly bind SNAPf tag; therefore, if labeling both SNAPf and CLIPf in the same specimen, we recommend doing sequential SNAPf substrate incubation (minimum 5 min) then addition of CLIPf substrate (minimum 5 min) to avoid cross-reactivity.