Lmx1a

Cells were rinsed in PBS and fixed in 4%PFA for 15 mins. Cells were then permeabilised via three washes in PBS containing 0.3% Triton-X-100 (PBST) and then blocked in PBST containing 1% BSA and 3% normal donkey serum. Primary antibodies were added in blocking solution for 2 hours at ambient temperature or overnight at 4 °C. The cells were washed in PBST three times before being incubated for 1 hour in the dark in Alexa-Fluor secondary antibodies, 1:200 (Invitrogen). Three PBST washes were then performed that included one with DAPI, 1:1000 (Molecular Probes). The primary antibodies used in this study were: FolR1(sheep, R&D), TH (rabbit, PelFreez), Pitx3 (rabbit, gift of M. Smidt, University of Amsterdam), Foxa2 (goat, Santa Cruz), Foxa2 (Rabbit, Abcam), Lmx1a (rabbit, gift of M. German, UCSF), Nestin (mouse, BD Pharmagen), GFAP (rabbit, Dako), GABA(rabbit, Sigma), 5HT(mouse, Abcam), Isl1(mouse, DSHB), Lim1/2(mouse, DSHB), Pax6(mouse, DSHB), Nkx6.1(mouse, DSHB), Dmrt5(rabbit, custom made). Images were taken on a Leica TCS SP5 confocal microscope. Quantification of markers was carried out manually by examining randomly selected fields from at least 3 independent experiments and presented as means ± sem.

Rosettes were stained with Nestin, Tuj-1, MAP2, TH, and LMX1A antibodies (all from Abcam, Cambridge, United Kingdom) after purification to check the purity of the NSCs. For immunofluorescence analysis of NSCs, cells were cultured on chamber slides, fixed with 4% PFA for 20 min, and permeabilized with 0.1% Triton X-100 for 15 min. They were then blocked with PBS containing 5% BSA for 1 h at room temperature. The samples were incubated overnight at 4 °C in blocking buffer (1% BSA) containing primary antibody (1:500). The details of antibodies showed in Table S1 and S2. The samples were washed three times with PBS overnight for 5 min each, and the cells were incubated with the corresponding secondary antibody (1:200) for 2 h at 37 °C. The samples were washed with the same wash protocol after secondary antibody incubation and stained with DAPI. Stained samples were examined with an FV1000 fluorescence microscope (OLYMPUS).