Af 488 donkey anti mouse

Immunofluorescent staining was performed to identify markers of neuronal differentiation as well as regional patterning. Coverslip staining was performed as previously described (Vermilyea et al.^{17 (link)}) using the primary antibodies (see Supplemental Table 4) against βIII-Tubulin, microtubule associated protein 2 (MAP2), tyrosine hydroxylase (TH), FOXA2, and PAX6 followed by the secondary antibody AF 488 donkey anti-mouse (A21202, Invitrogen) and Cy3 donkey anti-mouse (715-165-150, Jackson). Coverslips were then counterstained with phalloidin and/or DAPI. Images were taken with a Nikon A1 confocal microscope. For differentiation efficiency analysis, each data point represents a separate captured field.

Bovine serum albumin, Ponceau S, Sigmafast protease inhibitor cocktail tablets, Rosiglitazone and Insulin from Sigma. Primary antibodies: mouse anti-α tubulin clone B-5-1-2, mouse anti-acetylated tubulin clone 6-11-B-1, rabbit anti-γ tubulin and rabbit anti-laminin from Sigma; goat anti-human FSTL1, goat anti-mouse Fstl1, and mouse Fstl1 recombinant protein from R&D Systems, rabbit anti-BBS4 from ProteinTech, rabbit anti-LC3B from Cell Signaling Technology, mouse anti-human golgin-97 clone CDF4 from Invitrogen, and rabbit anti-calnexin, mouse anti-LAMP2 clone H4B4 and rat anti-BrdU from Abcam. Horseradish peroxidase (HRP)-conjugated secondary antibodies anti-mouse and anti-goat from Santa Cruz and anti-rabbit from Sigma. Alexa Fluor (AF)−594 donkey anti-goat, AF-488 donkey anti-mouse, AF-633 goat anti-rabbit, Alexa Fluor 488-conjugated anti-rat and tetramethylrhodamine goat-anti mouse were from Invitrogen. TRIzol reagent, Lipofectamine RNAiMAX and stealth RNAs were obtained from Invitrogen. Dexamethasone and 3-Isobutyl-1-Methylxanthine (IBMX) were obtained from AppliChem.