C1si fast spectral confocal system

The procedure for adult Drosophila nervous system dissection and subsequent dye-injection has been previously described in detail [55 (link), 56 (link)]. To visualize the morphology of the GF-TTMn connection either a 10 mM Alexa Fluor 555 Hydrazide (Molecular Probes) in 200 mM KCl or a dye solution of 10% w/v neurobiotin (Vector Labs) and tetramethylrhodamine isothiocyanate (TRITC)-dextran (Invitrogen) in 2 M potassium acetate was injected into the GF axons by passing hyperpolarizing or depolarizing current, respectively. Preparation of GF samples for confocal microscopy has been described previously [55 (link), 56 (link)]. The following antibodies and concentrations were used: Streptavidine-Cy3 conjugate (Jackson; 1:750 dilution), BP104 (Developmental Studies Hybridoma Bank, 1:50 dilution), goat anti-mouse-Cy2 and Cy5 (Jackson ImmunoResearch, 1:500 dilution), anti-GFP A11122 (Invitrogen, 1:500 dilution). Samples were scanned at a resolution of 1024x1024 pixels, 2.5x zoom, and 0.5 μm step size with a Nikon C1si Fast Spectral Confocal system using a 60×/1.4 NA oil immersion objective lens. Images were processed using Nikon Elements Advance Research 4.4 and Adobe Photosuite CS5 software.

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)}.