Examination of stained embryos using a dissecting stereomicroscope. (A) Antibody-stained Drosophila embryos in a pool of 70% glycerol on a standard microscope slide. Embryos are 0–24 h old and collected from a robo1¹/CyO,wg-lacZ stock, stained with mAb 1D4 (anti-FasII) and anti-β-gal primary antibodies and an horseradish peroxidase (HRP)-conjugated secondary antibody, and then developed with stable DAB immunohistochemistry. Stained embryos can be examined under a standard dissecting stereomicroscope and sorted for stage and genotype. (B) β-gal-positive embryos (identified by the presence of staining in segmental stripes characteristic of the wg gene) carry the CyO,wg-lacZ chromosome and thus are heterozygous for the robo1¹ allele (robo1¹/+). Note that the motor neurons project laterally in every segment, but axons are readily distinguishable from the stripes of wingless positive cells under low magnification. (C) Embryos that lack β-gal expression are homozygous for the robo1¹ allele (robo1¹/robo1¹). Anti-FasII staining is present in both populations (B,C). β-gal-positive heterozygous embryos exhibit wild-type arrangement of FasII-positive axon pathways, whereas their β-gal-negative homozygous mutant siblings exhibit ectopic midline crossing characteristic of robo1 mutants. Older embryos (late stage 17) exhibit weaker staining because of the formation of cuticle that interferes with antibody penetration.