Quemasa

Third instar larvae were dissected in fresh Ca²⁺ free HL3 (110 mM NaCl, 5 mM KCl, 10 mM NaHCO₃, 5 mM Hepes, 30 mM sucrose, 5 mM trehalose, and 10 mM MgCl₂, pH 7.2;^{87 (link)}), nerves were cut and larvae subsequently incubated for 10 min in HL3 with 1.5 mM CaCl₂ and 60 mM KCl. Multiple steps of washing with HL3 before fixation removed the non-internalized dye. Larvae were fixed in 4% paraformaldehyde (Laborimpex, 15714) and 2.5% glutaraldehyde (Polysciences, Inc, 111-30-8) in 0.1 M sodium-cacodylate buffer pH 7.4 (Merck, C0250-500g) at 4 °C for at least 24 h. The next day the larvae were osmicated in 2% osmium tetroxide (Laborimpex AGR1023) for 2 h on ice. After staining in 2% aqueous uranyl acetate solution (EMS #22400) for at least 1.5 h and dehydration in an ascending series of ethanol solutions, the samples were embedded in Agar 100 (Laborimpex, AGR1031) and cured at 60 °C for 48 h. Ultrathin sections (70 nm) were cut with a Dumont Diamond Knife on a Leica UCT ultra-microtome and collected on copper grids (Van Loenen, 01805-F) and imaged on a JEM 1400 transmission electron microscope (JEOL) at 80 kV with a bottom mounted camera (Quemasa; 11 megapixels; Olympus) running iTEM 5.2 software (Olympus). Statistical analyses were performed on larvae from 3 independent crosses per genotype. More than 5 NMJs per larvae were imaged.

Adult fly heads were dissected and immediately fixed in 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M Na-Cacodylate buffer (pH 7.4) for 2 h at room temperature. Samples were further fixed at 4 °C overnight, and then washed with 0.1 M Na-Cacodylate, pH 7.4, and subsequently osmicated with 2% osmium (OsO₄/Na-Cacodylate buffer). After dehydration in an ascending series of ethanol solutions and staining in 4% aqueous uranyl acetate solution the specimen were embedded in Agar 100 (Laborimpex; Agar Scientific). Ultrathin sections (70 nm) of the retina and the lamina were collected on grids (Laborimpex; Agar Scientific) coated with Butvar and imaged on a JEM 1400 transmission electron microscope (JEOL) at 80 kV with a bottom mounted camera (Quemasa; 11 megapixels; Olympus) running iTEM 5.2 software (Olympus).