Whole-Mount Immunostaining of Zebrafish Embryos

Embryos were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 2–3 h at room temperature or overnight at 4 °C. Whole-mount antibody staining was performed using the following antibodies: anti-DeltaA (18D2, ZIRC) (1:50); anti-DeltaC (zdc2, Abcam, UK) (1:500); anti-DeltaD (zdd2, Abcam, UK) (1:500); anti-Scl (ref. 43 (link)) (1:50); anti-mCherry (ab167453, Abcam, UK) (1:500); and anti-RFP (8D6, MBL, Japan) (1:500). Goat anti-mouse IgG Alexa568 (A-11031, Thermo Fisher Scientific, USA) (1:1,000) and goat anti-rabbit IgG Alexa546 (A-11035, Thermo Fisher Scientific, USA) (1:1,000) were used as the secondary antibodies. The Histofine Simple Stain MAX PO (R) (Nichirei Bioscience, Japan) and the TSA Cyanine 5 System (PerkinElmer, USA) were also used for Scl detection.