Cy3 donkey anti sheep

Embryos were fixed overnight at 4°C in 4% paraformaldehyde (Santa Cruz #sc-281692) dissolved in PBS (1x phosphate buffered saline pH 7.5). Embryos were then washed 6x with PBST (PBS with 0.2% Tween; Sigma #P1379) for 10 min/each and placed in blocking solution (PBST with 1% bovine serum albumin; Sigma #A8022) for 1 h. Embryos were then incubated overnight at 4°C with primary antibodies diluted in blocking solution and then washed with PBST 6x for 10 min/each. Embryos were then incubated overnight at 4°C with fluorescent secondary antibodies diluted in blocking solution for 2 h at RT or overnight at 4°C. Embryos were then washed in PBST 4x for 15 min/each and mounted in an aqueous 0.5% agarose solution before imaging. Primary antibody dilutions used as follows. To label somite boundaries, mouse anti-pFAK Tyr397 (Millipore #05–1140 at 1:1000); to enhance the visualization of EGFP, chicken anti-GFP (Invitrogen #A10262 at 1:1000); to improve mCherry detection, sheep anti-mCherry (custom-made at 1:1000; a gift from Holger Knaut). The following secondary antibody dilutions were used: Alexa Fluor 488 goat anti-chicken (Invitrogen #A11039 at 1:1000), Cy3 donkey anti-sheep (Jackson ImmunoResearch #713-165-147 at 1:1000), and Alexa Fluor 647 donkey anti-mouse (Invitrogen #A31571 at 1:1000).

At the conclusion of experiments (6–8 weeks after viral injection), mice were first anesthetized with a ketamine (100 mg/kg) and xylazine (7 mg/kg) mixture then perfused with 4% paraformaldehyde (Thermo Fisher Scientific), and the brains were extracted and cryoprotected in 30% sucrose in 1× PBS until they sank. Sections of 40 µm were taken in a cryostat and stored in 1× PBS at 4 °C until they were used for immunohistochemistry experiments. The following antibodies and dilutions were used: chicken polyclonal anti-GFP (1:500, Abcam, ab13970), Cy3 donkey anti-sheep (1:200, Jackson ImmunoResearch, 713-165-147), sheep polyclonal anti-tyrosine hydroxylase (1:1000, Abcam, ab113), Cy2 donkey anti-chicken (1:200, Jackson ImmunoResearch 703-225-155).