Fitc conjugated goat anti hrp

Drosophila embryo collection, fixation, and antibody staining were carried out as previously described (Hauptman et al., 2022 (link); Patel, 1994 (link)). The following antibodies were used: FITC-conjugated goat anti-HRP (Jackson ImmunoResearch #123–095-021, 1:100), mouse anti-Fasciclin II (Developmental Studies Hybridoma Bank [DSHB] #1D4, 1:100), mouse anti-βgal (DSHB #40–1a, 1:150), mouse anti-Robo3 cytoplasmic (DSHB #15H2, 1:100), mouse anti-HA (Covance #MMS-101P-500, 1:1000), Cy3-conjugated goat anti-mouse (Jackson #115–165-003, 1:1000). Embryos were genotyped using balancer chromosomes carrying lacZ markers. Ventral nerve cords from embryos of the desired genotype and developmental stage were dissected and mounted in 70% glycerol/PBS. Fluorescent confocal stacks were collected using a Leica SP5 confocal microscope and processed by Fiji/ImageJ (Schindelin et al., 2012 (link)) and Adobe Photoshop software.

Wandering third instar larvae were collected and dissected in ice-cold Ca²⁺-free HL3 saline. Larval fillets were fixed for 20 min in 4% formaldehyde in phosphate-buffered saline (PBS). Antibody staining for fixed samples was performed as previously described (Nahm et al., 2010a (link)). The following primary antibodies were used in this study: mouse anti-FasII (1D4; DSHB, USA) at 1:20 and FITC-conjugated goat anti-HRP (Jackson ImmunoResearch Laboratories, USA) at 1:200. A Cy3-conjugated donkey anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, USA) was used at a dilution of 1:200. Confocal images were taken with an Olympus FV300 laser-scanning confocal microscope using a Plan Apo 40× 0.90 NA or U Plan Apo 100x 1.35 NA objective and processed using FLOUVIEW imaging analysis software. To compare different genotypes, samples for each experiment were prepared simultaneously and imaged under identical confocal settings.
Bouton number was quantified at NMJ 6/7 in abdominal segment 2 after anti-HRP staining as previously described (Nahm et al., 2010a (link)). For quantification of synaptic FasII levels, larval NMJs were double-stained with anti-HRP and anti-FasII antibodies. The fluorescence intensity of synaptic FasII was measured using ZEN imaging software (Carl Zeiss, Germany) and then normalized to HRP intensity.