Rabbit anti wnt5a

On day 65 of differentiation, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization solution 1 containing 0.1% (v/v) x-100, 0.1% (v/v) Tween-20, 20% (v/v) Hybri-Max dimethylsulphoxide (DMSO) in DPBS without calcium or magnesium (DPBS −/−). Then further incubated with permeabilization solution 2 containing 0.1% (v/v) Tween-20, 0.1% (v/v) Triton X-100, 0.1% deoxycholate, 0.1% tergitol solution, 20% (v/v) DMSO in DPBS (−/−). Cells were blocked with 3% (v/v) donkey serum in permeabilization solution 2 following incubation with primary antibody rabbit anti-PITX3 (1:100 Invitrogen), sheep anti-TH (1:500 Abcam), goat anti-GIRK2 (1:100 Abcam), rabbit anti-WNT5A (1:200 Abcam), mouse anti-TUBB3/chicken anti-MAP2 (1:1000 Abcam) in 3% donkey serum (1:100 dilution) overnight at 4°C. Then cells were stained with a secondary antibody (Supplementary Table S1) in 3% donkey serum (1:1000 dilution) for 4 h at room temperature. On the day of imaging, cells were stained with DAPI (1:5000 dilution) in DPBS (−/−). Fluorescence was detected using a Nikon A1R Confocal Microscope.