Zebrafish Immunofluorescence: Detailed Protocol

Zebrafish samples were prepared for immunofluorescence as previously described (Fausett and Goldman, 2006 (link); Ramachandran et al., 2010a (link); Ramachandran et al., 2010a (link)). Primary antibodies used in this study: anti-pSmad3, Abcam Cat. # ab52903 (1/200); Zpr-1 and Zn-5, Zebrafish International Resource Center (1/500 and 1/1000, respectively); anti-HuC/D, Abcam, Cat. #ab210554 (1/500); anti-PKC_β1, Santa Cruz Biotechnology, Cat. #SC-8049 (1/200); anti-glutamine synthetase (GS), Sigma-Aldrich, Cat. #MAB302 (1/500); anti-SOX9, EMD Millipore, Cat. #AB5535 (1/500); anti-BrdU, Thermo Fisher, Cat. # MA 1–82088 (1/500) and Cat. # B35128 (1/500, clone MoBu-1 for co-staining with EdU Click-It chemistry). Secondary antibodies: Alexa Flour 555 Donkey anti Mouse-IgG (H+L), Thermo Fisher Cat. # A31570 (1:500); Alexa flour 555 Donkey anti Rabbit IgG (H+L), Thermo Fisher, Cat # A31572 (1:500); Alexa flour 555 Donkey anti Sheep IgG (H+L) Thermo Fisher Cat #A21436. Cy3, Jakson Immuno research labs, Cat #712-166-150 (1:500); Alexa Flour 488 donkey anti mouse Thermo Fisher Cat. # A21202 (1:500); Alexa Flour 488 goat anti rabbit Thermo Fisher Cat. # A11008 (1:500); Cy5 goat anti mouse, Thermo Fisher Cat. # A10524 (1:500); and Alexa Flour 647 goat anti rabbit Thermo Fisher Cat # A21244 (1:500). In situ hybridization was performed as described previously (Barthel and Raymond, 2000 (link)).