Cells were fixed with 4% PFA for 10 min, washed with PBS, and blocked with 0.2% Triton X in PBS supplemented with 5% BSA and 10% goat/donkey serum. Cells were then incubated with primary antibodies in 0.2% Triton X in PBS with 5% BSA and 5% goat/donkey serum: rabbit anti-LMX1A (1:2,000, Millipore), goat anti-FOXA2 (1:50; Santa Cruz), rabbit anti-TH (1:500; Pel-Freez), mouse anti-TUJ1 (1:2,000; Promega), rabbit anti-TUJ1 (1:2,000; Covance), rabbit anti-OTX2 (1:1000; Millipore), mouse anti-Nestin (1:50; R&D), rabbit anti-BF1 (1:100; Abcam), mouse anti-NKX2.1 (1:200; Chemicon), rabbit anti-PAX6 (1:300; Covance), rabbit anti-Glutamate (1:2000; Sigma), rabbit anti-VGLUT1 (1:500; Synaptic Systems), rabbit anti-VGLUT2 (1:500; Synaptic Systems), mouse anti-GABA (1:100, Sigma), and rabbit anti-VGAT (1:500; Synaptic Systems).
Corresponding fluorescent-labeled secondary antibodies were used (Alexa-Fluor 488 for green, Alexa-Fluor 555 for red; R&D). Images were captured using a Carl Zeiss Axiovert 200M (Jena, Germany) microscope.
Corresponding fluorescent-labeled secondary antibodies were used (Alexa-Fluor 488 for green, Alexa-Fluor 555 for red; R&D). Images were captured using a Carl Zeiss Axiovert 200M (Jena, Germany) microscope.