Drosophila Embryo Immunostaining Protocol

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.