Immunostaining of Kupffer's Vesicle Cilia

Embryos were fixed at the 8-somite stage using 4% paraformaldehyde at 4°C overnight. Anti-acetyl-alpha-tubulin antibody (1:500, Sigma, MABT868, USA) was used to label the cilia in Kupffer's vesicles (KVs). Immunofluorescence staining was performed as previously described (33 (link)). Before permeation with acetone, embryos were equilibrated in Tris–HCl (0.1 M, pH 9.0) for 5 min at room temperature and then heated at 70°C for 15 min for antigen retrieval. After staining, embryos were flattened and imaged using the z-stack function of the confocal microscope with a ×60 oil objective.