Embryos were fixed in 4% paraformaldehyde (2–8 hrs, dependent on developmental age), cryopreserved in 20% sucrose and coronally sectioned (16 μm, 1:10 series). For primary cultures and explant assays, cells were fixed with 4% paraformaldehyde, rinsed and stored in PBS with 0.03% sodium azide.
Immunohistochemistry was performed using previously described methods21 (link). Antibodies used were as follows: goat anti-CHL1 (1:500, R&D Systems), rabbit anti-NURR1 (1:200, Santa Cruz), rabbit anti-TH (1:800, PelFreez), chicken anti-TH (1:400, Abcam), mouse anti-TUJ1 (1:15000, Promega), and chicken anti-TUJ1 (1:200, Millipore). Appropriate secondary antibodies, donkey 488, 555 and 647 Alexaflour (Jackson ImmunoResearch Laboratories), were used at a dilution of 1:400. Images were captured using fluorescence microscope (Zeiss Axio Observer Z1 or Zeiss 780 confocal microscrope).
Immunohistochemistry was performed using previously described methods21 (link). Antibodies used were as follows: goat anti-CHL1 (1:500, R&D Systems), rabbit anti-NURR1 (1:200, Santa Cruz), rabbit anti-TH (1:800, PelFreez), chicken anti-TH (1:400, Abcam), mouse anti-TUJ1 (1:15000, Promega), and chicken anti-TUJ1 (1:200, Millipore). Appropriate secondary antibodies, donkey 488, 555 and 647 Alexaflour (Jackson ImmunoResearch Laboratories), were used at a dilution of 1:400. Images were captured using fluorescence microscope (Zeiss Axio Observer Z1 or Zeiss 780 confocal microscrope).