Whole mount in situ hybridization was performed essentially as described (36 (link)). Embryos underwent overnight fixation in 4% paraformaldehyde at 4°C. After dehydration, bleaching and rehydration, in situ hybridization was performed with DIG-labeled antisense riboprobes for Kit, Dct and MITF. All steps except probe hybridization and final colorimetric detection were performed on a Biolane HTI (Hölle & Hüttner AG, Tübingen, Germany).
Immunohistochemistry was performed on 10-μm-thick sections from the trunk of embryos after fixation, embedding, freezing and cryotome sectioning using the following primary antibodies: anti-Islet1 mouse monoclonal (1:500 dilution, Developmental Studies Hybridoma Bank), anti-MITF rabbit antiserum (1:1000 dilution, gift of H. Arnheiter, NIH, Bethesda, MD, USA), anti-FABP7 rabbit antiserum (1:300 dilution; Millipore), anti-SOX10 guinea pig antiserum [1:1000 dilution (37 (link))]. Secondary antibodies conjugated to Cy2, Cy3 (1:200 dilution, Dianova) or Alexa488 (1:500 dilution, Molecular Probes/Invitrogen) immunofluorescent dyes were used for detection. Nuclei were counterstained with 4΄,6-diamidin-2-phenylindole (DAPI).
β-Galactosidase (β-gal) staining was performed on whole embryos as previously described (32 (link)).
Immunohistochemistry was performed on 10-μm-thick sections from the trunk of embryos after fixation, embedding, freezing and cryotome sectioning using the following primary antibodies: anti-Islet1 mouse monoclonal (1:500 dilution, Developmental Studies Hybridoma Bank), anti-MITF rabbit antiserum (1:1000 dilution, gift of H. Arnheiter, NIH, Bethesda, MD, USA), anti-FABP7 rabbit antiserum (1:300 dilution; Millipore), anti-SOX10 guinea pig antiserum [1:1000 dilution (37 (link))]. Secondary antibodies conjugated to Cy2, Cy3 (1:200 dilution, Dianova) or Alexa488 (1:500 dilution, Molecular Probes/Invitrogen) immunofluorescent dyes were used for detection. Nuclei were counterstained with 4΄,6-diamidin-2-phenylindole (DAPI).
β-Galactosidase (β-gal) staining was performed on whole embryos as previously described (32 (link)).
