Zen 2.1 acquisition software

The localization of the transgenic synaptotagmin protein was visualized by immunohistochemistry. L3s were dissected in Ca²⁺-free HL3.1, fixed in PBS containing 4% formaldehyde for 1 hour, incubated with a 1:400 dilution of Dsyt-CL1 in dilution media [PBS with 0.1% Triton (PBST), 1% BSA, and 1% NGS] overnight at 4°C, washed in PBST for 1–3 hours, incubated in dilution media containing a 1:400 dilution of Alexa Fluor 488 goat anti-rabbit antibody (Invitrogen, Carlsbad, CA) for 1 hour at room temperature, washed in PBST for one hour, and mounted on microscope slides in Citifluor (Ted Pella, Redding, CA). Confocal images of the neuromuscular junction on muscle fibers 6 and 7 were taken on a Zeiss 880 light-scanning microscope (Zeiss, White Plains, NY), with a 40x objective and Zeiss Zen 2.1 acquisition software, version 11.0.3.190.

For immunolabeling of the neuromuscular junction, third instars of P[syt^WT] heterozygotes and P[syt^P-L] heterozygotes were dissected in Ca²⁺-free HL3.1 saline to expose their body wall muscles and fixed in phosphate-buffered saline (PBS, 137 mM NaCl, 1.5 mM KH₂PO₄, 2.7 mM KCl, 8.1 mM Na₂HPO₄) containing 2% formaldehyde for 1 hour. Whole-mounts were incubated overnight in Dsyt-CL1 diluted 1:400 in dilution media [PBS with 0.1% Triton, 1% bovine serum albumin, and 1% normal goat serum (NGS from Jackson ImmunoResearch, West Grove, PA)], washed in PBST (PBS with 0.1% Triton) for 3 hours, incubated in Alexa Fluor 488 goat-anti-rabbit antibody (Invitrogen, Carlsbad, CA) diluted 1:400 and Texas Red anti-HRP (Jackson ImmunoResearch, West Grove, PA) diluted 1:50 in dilution media for 1 hour, washed in PBST for 1 hour, and mounted in Citifluor (Ted Pella, Redding, CA). To label transgenic synaptotagmin expressed in the syt^null background, the above protocol was applied to first instars rather than third instars with the following changes: first instars were fixed in PBS containing 4% formaldehyde for 2 hours and Texas Red anti-HRP was omitted. Neuromuscular junctions at the muscle 6/7 junction were imaged for both first and third instars with a Zeiss 880 light scanning microscope using a 40X objective, and acquired using Zeiss Zen 2.1 acquisition software, version 11,0,3,190.