As 2024

For whole-mount immunohistochemistry, larvae were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) overnight at 4°C. Larvae were then washed three times in PBS with 0.25% Triton X-100 (PBT) and permeabilized in 0.05% trypsin-EDTA on ice. After washing, samples were blocked with 2% donkey serum for 1 hour at room temperature and incubated overnight at 4°C with primary antibodies diluted in PBT + 1% bovine serum albumin and 1% dimethyl sulfoxide. Larvae were then washed in PBT and incubated with secondary antibodies at room temperature for 2 to 4 hours. Primary antibodies used were rabbit anti–red fluorescent protein (1:500; 632496, Clontech), chicken anti-GFP (1:500; ab13970, Abcam), goat anti-WGA (1:500; AS-2024, Vector Labs), sheep anti–Clostridium botulinum BoNT-B Light Chain antibody (1:500; AF5420, R&D Systems), rabbit anti-NR1 (1:200; 114011, Synaptic Systems), and anti-synaptophysin [1:200; S5768, Sigma-Aldrich (62 (link))]. After staining, the lower jaw was carefully removed under a dissecting microscope, and fish were mounted and imaged from the ventral side using a confocal microscope (Leica SP8-UV).