Whole-mount Immunostaining Protocol for Zebrafish Embryos

Whole-mount immunostaining was performed as previously described with slight modification [40 (link)]. For Phospho-smad 1,5,9 antibody staining, embryos were fixed in 4% PFA, washed in PBS, boiled in sodium citrate solution for 2 minute and heated at 60°C for an hour for antigen retrieval. Embryos were blocked and incubated in primary antibody against Alcama (Zn5, Zebrafish International Resource Center) at a dilution of 1:250, Phospho-smad 1,5,9 (Cell Signaling, catalog no. 9511) at a dilution of 1:100, MF20 (Hybridoma bank) at a dilution of 1:50 or β-catenin (Sigma, catalog no. C7207) at a dilution of 1:500. Texas red-conjugated anti-mouse secondary antibody and Alexa Fluor 488 conjugated anti-rabbit secondary antibody was used at 1:100 dilution (Molecular probes/Invitrogen). Texas red or Alexa Fluor 647 conjugated phalloidin (Molecular Probes/Invitrogen) was used 1:100 dilution. Alexa Fluor 555 conjugated Wheat Germ Agglutinin (WGA, Molecular Probes), a lectin that binds to N-acetyl glycosamino glycan, was used at 1μg/ml concentration. TO-PRO-3 (Molecular Probes) was used at 1:1000 dilution for nuclear counterstain. To stain larvae and juvenile fish, hearts were dissected before incubating in the staining solution.