Characterization of zebrafish anti-Dvl2 antibody. (A) Alignment of zebrafish Dvl 1,2 and 3 along with human DVL2 (hum DVL2) epitope region and surrounding conserved nucleotides. The number 573 refers to the amino acid position in zebrafish Dvl2, the first amino acid of the epitope, which is overlined. (B) Western blot analysis of zebrafish embryos injected with Dvl2^HA mRNA (+) or uninjected (-) and probed with anti-Dvl2 antibodies at 1:1000 or 1:20,000. The pre-immune serum (pre) did not detect any zebrafish proteins. The predicted molecular weight of Dvl2 is 81.8 kDa and with the HA epitope tag is predicted to be 82.9 kDa. The * denotes a nonspecific band. (C, D) Western analysis of Dvl2 protein at 1 day postfertilization in the presence of increasing amounts (0, 2, 4, and 6 ng) of Dvl2 antisense MO (lanes 1, 2, 3, and 8) or co-injected with Dvl2^HA mRNA that is sensitive to the MO (lanes 4, 5, 6, and 7). The adjusted levels of Dvl2 were plotted (D) relative to uninjected embryos (lane 8 in C). (E, F) One blastomere of an eight-cell stage embryo was injected with Dvl2^HA mRNA, fixed at 4.7 hpf (30% epiboly), and incubated with anti-HA (1:200) (E) or anti-Dvl2 (1:5000) (F) antibodies. Uninjected embryos have robust and ubiquitous Dvl2 staining at 1:1000 (G). The anti-HA and anti-Dvl2 (1:5000) immunostaining give very similar results. (H–J) Colabelling of mosacially overexpressed Dvl2^HA, similar to that described in E. Scale bar = 20μM. hpf, hours postfertilization.