Ab10533

Cells were fixed with 4% paraformaldehyde for 30 min at room temperature, and then washed with 0.1% Tween-20/PBS, 5 min, twice at room temperature. Cells were permeablized with 1% TritonX/PBS for 30 minutes at room temperature and blocked with 4% donkey serum/PBS, 30 min, room temperature. The primary antibodies were diluted in blocking solution and incubated with cells overnight at 4 °C, and then the cells were washed with 0.1% Tween-20/PBS, 5 min, three times at room temperature. Secondary antibodies were diluted in PBS at 1:200 dilution, incubated with processed cells for 1 hour at room temperature, and then washed with 0.1% Tween-20/PBS, 5 min each, three times at room temperature. Counterstaining was accomplished with DAPI/PBS (1 ug/mL DAPI in PBS). Primary antibodies were LMX1A (Millipore, ab10533), FOXA2 (Millipore, ab4125), OTX2 (Millipore, ab9566), TH (Pel Freez, P40101), GIRK2 (Abcam, ab30738), Nurr1 (Santa Cruz, sc-990) and TUBB3 (Covance, MRB-435 P). Secondary antibodies were from donkey (Jackson ImmunoResearch laboratories).

Cells were fixed in 4% of paraformaldehyde in Phosphate Buffer Saline (PBS), blocked for 30 min in PBS supplemented with 5% of normal donkey serum and 0.3% of triton X-100. Primary antibodies used were directed against FoxA2 (sc-6554, 1/100, Santa Cruz,); Ki67 (550609, 1/250, BD); Lmx1a (AB10533, 1/250, Millipore); Map2 (M2320, 1/500, Sigma); Nanog (AF1997, 1/250, R&F), Nestin (NB100-1604, 1/500, Novus Biologicals); Oct4 (sc-5279, 1/500, Santa Cruz); Pax6 (AB-528427, 1/1000, DSHB); Sox1 (AF3369, 1/500, R&D); TH (CH23006, 1/500, Acris Antibodies GmbH); Tra-1-81 (MAB4381, 1/500, Millipore); or βIII tub (MMS-435P, 1/1500, Covance). Nuclei were counterstained with Dapi (1/1500). Immunocytochemistry preparations were imaged using a Nikon A1 confocal microscope.

The antibodies to EMX2 (ab94713; 1:50), FOXG1 (ab18259; 1:400), p-VIM (ab22651; 1:120), SOX2 (ab79351; 1:500), SATB2 (ab51502; 1:50), CTIP2 (ab18465; 1:250), TBR1 (ab31940; 1:500) and CUX1 (ab54583; 1:200) were from Abcam. The antibodies to OCT3/4 (sc5279; 1:22) and LIFR (sc-515337; 1:100) were from Santa Cruz. The antibody to PAX6 (supernatant, 1:22) was from DSHB. The antibodies to SOX1 (AF3369; 1:40), SOX2 (AF2018; 1:100) and OLIG3 (MAB32456; 1:450) were from R&D. The antibodies to NR2F1 (ABE1425; 1:500), DCX (AB2253; 1:500) and LMX1A (AB10533; 1:1,000) were from Millipore. The antibodies to SP8 (HPA054006; 1:50) and MEF2C (HPA005533; 1:100) were from Atlas Antibodies. The antibodies to CASP3 (cat. no. 9661; 1:500) and TCF7L2 (cat. no. 2569; 1:500) were from Cell Signaling. The antibody to TTR (AHP1837; 1:500) was from Bio-Rad. The antibodies to HOPX (HPA030180; 1:500), PTPRZ1 (HPA015103; 1:500) and EMX1 (HPA006421; 1:50) were from Sigma-Aldrich. Secondary Alexa Fluor antibodies 488, 546 and 647 (1:700) were obtained from Invitrogen.