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Fig. 4 Zebrafish embryos expressed integrin αVβ3 at 48 hpf but not at 24 hpf. (A) The 48-hpf zebrafish embryos were fixed and incubated with the LM609 antibody. LM609 immunoreactivity was revealed using a rhodamine-labeled fluorescent secondary antibody and preparations were examined using confocal imaging. At 48 hpf, LM609 immunoreactivity specifically labeled cells along the dorsal spinal cord (asterisks), the location of Rohon-Beard cells (RBs). LM609 also labeled cells in the ventral spinal cord (arrowheads), consistent with the location of motoneurons. (B) In contrast, 24-hpf embryos did not display LM609 immunoreactivity, consistent with an absence of integrin αVβ3 expression at 24 hpf. (C) Pre-incubation of LM609 with 50 μg/ml αVβ3 protein blocked immunoreactivity in 48 hpf embryos, indicating that the labeling was specific for αVβ3. (D) Tg(isl3:GFP) transgenic zebrafish embryos expressed green fluorescent protein (GFP) in RBs under control of the islet3 promoter. (E) LM609 immunoreactivity was present in dorsal cells. (F) GFP and LM609 immunoreactivity colocalized in RB soma and axon tracts. Scale bars: 200 μm (A-C); 20 μm (D, E, F).