Fig. S4

Flt1 immunohistochemistry. (A) Flt1 immunostaining (red) with tyramide signal amplification (TSA) in Tg(fli:egfp)^y1 embryos. Note Flt1 immunostaining in the vessel and somite. (B) Flt1 immunostaining (red) in Tg(fli:egfp)^y1 embryos in controls (left), after 3 ng flt1 ATG MO (middle) and after 6 ng flt1 ATG MO (right). Note the reduced immunostaining after MO-mediated knockdown of flt1. (C) Negative controls for immunostaining with the custom-made antibody against zebrafish Flt1 protein. Background staining was low in all three negative control immunostaining conditions. (Left) IgG, primary antibody (Ab) was against rabbit IgG. (Middle) Flt1Ab+peptide, embryos were incubated with primary antibody against Flt1 and the protein epitope used to generate the zebrafish Flt1 antibody prior to performing immunostaining. (Right panel) 2^nd Ab, primary antibody omitted and stained with secondary antibody only. (D) Quantification of neuronal numbers in Tg(flt1^BAC:yfp) × Tg(kdrl:ras-cherry) controls and flt1 morphants (percentage of control). Embryos were evaluated at 48 hpf. Error bars indicate s.e.m.