Anti nsf

Antibodies and their staining protocols used in this research were described previously.^{13 (link)} Briefly, after 5 dpf zebrafish larvae were fixed in PBS buffer supplied by 4% PFA/4% Sucrose/0.01% Tween-20 overnight at 4° C, they were then permeabilized with acetone for 7 minutes at −20°C and blocked in PBS containing 2% fish gelatin/1% BSA/1% goat serum/1% DMSO (FGBS). After blocking, specimens were incubated with primary antibodies in FGBS overnight at 4° C (1:100 anti-NSF, Cell Signaling Technology; 1: 4000 anti-Ribeye b, Openbiosystems; 1:500 anti-zn12/HNK-1, Zebrafish International Resource Center; 1:500 anti-pan MAGUK, UC Davis/NIH NeuroMab Facility; 1: 1000 Vglut3, Proteintech; 1:50 anti-Mbp, gift from W. Talbot; 1:1500 anti-Acetylated tubulin, Sigma; 1:1000 anti-FIGQY, gift from M. Rasband), followed by incubation with appropriate secondary antibodies (1:1500 conjugated to Alexa 488 or Alexa 568, Invitrogen) in FGBS overnight at 4° C. A Zeiss Axiovert ImagerM.1 microscope with an LSM700 confocal scanhead, Axiocam MrM camera, and water-immersion lens Zeiss Plan Apochromat 63X/1.4NA objective (Zeiss) were used to take Z-stack images of the lateral line nerve and neuromasts.

Antibodies used for western blotting were as follows: anti-Flag M2 (1:10000, Sigma-Aldrich, St. Louis, MO, USA); anti-NSF (1:500, Cell Signaling, Danvers, MA, USA); anti-LRRK2 (1:1000, C41-2, Abcam, Cambridge, UK); anti-Synaptobrevin, anti-synaptophysin and anti-Synaptotagmin 1 (1:1000, Synaptic System, Göttingen, Germany).
Between 10 and 20 μg of protein samples were dissolved in 4–20 % Tris-glycine polyacrylamide gels (Biorad) in SDS/Tris-glycine running buffer. Precision Plus molecular weight markers (Biorad) were used for size estimation. Solubilized proteins were then transferred to polyvinylidenedifluoride (PVDF) membranes in transfer buffer containing 10 % methanol. The PVDF sheets were blocked in Tris-buffered saline plus 0.1 % Triton (TBS-T) plus 5 % nonfat dry milk for 1 h at 4 °C and then incubated overnight at 4 °C with primary antibody in TBS-T plus 5 % non-fat dry milk. The PVDF membranes were washed in TBS-T (3 × 10 min) at room temperature (RT) followed by incubation for 1 h at RT with horseradish peroxidase-conjugated anti-mouse IgG. Blots were then washed in TBS-T (4 × 10 min) at RT and rinsed in TBS, and immunoreactive proteins were visualized using enhanced chemiluminescence plus (ECL+, GE Healthcare, Waukesha, WI, USA). Densitometric analysis was carried out using Image J software.