Dye Injection and Immunohistochemistry of Drosophila Nervous System

Dye injection and immunohistochemistry methods have previously been described in detail^{62 (link),63 (link)}. In brief, the ventral nerve cord of adult Drosophila was dissected and mounted dorsal side up on VECTABOND™ (Vector Labs) coated 0.9–1.1 mm etched slides. An 80–100 MΩ glass electrode filled with a dye solution of 10% w/v neurobiotin (Vector Labs) and tetramethyl rhodamine-labeled dextran (Invitrogen) and backfilled with 2 M potassium acetate was used to inject the dyes into the GF axons by passing depolarizing current. Samples were fixed in 4% paraformaldehyde and were prepared for confocal microscopy as described previously^{62 (link),63 (link)}. Streptavidin-Cy2 conjugate (Jackson ImmunoResearch; 1:750) was used to visualize neurobiotin. Samples were scanned at a resolution of 1024 × 1024 pixels, 2.5× zoom, and 0.5 μm step size with a Nikon C1si Fast Spectral Confocal system using a 60× oil immersion objective lens. Dye filling of the GFs with Lucifer Yellow was performed as described in ref. ^{15 (link)}.