Bp104

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.

We used image stacks of the Janelia database of the larval Drosophila CNS (wandering third instar; data by courtesy of co-author JWT). Data recording followed the protocol described in Li et al. (2014 (link)): The employed labels were “mouse antineuroglian (1:50 BP-104; Developmental Studies Hybridoma Bank), rat anti-N-cadherin (1:50 DN-Ex #8; Developmental Studies Hybridoma Bank) and rabbit anti-GFP immunoglobulin (Ig) G (1:1,000; Invitrogen A11122)”. The size of the 8 bit image stacks is about 1000 × 1400 × 100 voxels (in-plane resolution: 0.5 μm, slice thickness: 2 μm, z-step: 2 μm).