Commissural neurons were cultured for 24 hr in vitro and subsequently stimulated with VEGF (10 or 25 ng/ml, R&D systems, #493-MV) for 30 min. For immunostaining, neurons were fixed in 4% PFA/4% sucrose (complemented with proteinase and phosphatase inhibitors [Roche]) for 15 min at room temperature. Immunostaining for P-SFK was performed using a Rabbit (polyclonal) anti-Src (pY418) phosphorylation site specific antibody (Invitrogen, #44660G) followed by an Alexa-488 conjugated secondary antibody. For immunoblotting,

neurons were lysed in RIPA buffer complemented with proteinase and phosphatase inhibitors (Roche). An anti-Phospho-Src Family antibody (Cell Signaling, #2101) was used to probe the western blots. Subsequently blots were stripped and reprobed with an anti-Src Dabrafenib solubility dmso (36D10) antibody (Cell Signaling, #2109). The average of the phospho-SFK Selleckchem BMS-754807 fluorescence signal was measured for each growth cone using Image J and normalized to the average fluorescence signal in control growth cones. At least 50 growth cones were analyzed in two independent experiments (performed in triplicates) and statistical differences were assessed by unpaired t test versus control conditions. Floor plates (FPs) isolated from E11.5 mouse embryos were cultured in

three dimensional rat tail collagen in B27-supplemented Neurobasal medium. Conditioned medium from FPs (explants from a single FP were cultured in 300 μl) or control medium were collected after 48 hr and processed for further measurements

of VEGF and Shh protein concentration using the commercial Quantikine human VEGF ELISA kit (R&D Systems) and Shh ELISA kit (Abcam, ab100639), respectively. Flk1 protein expression was determined in lysates of E13 rat dorsal spinal cord tissue using the commercial mouse Flk1 ELISA kit (R&D Systems, Quantikine MVR200B). Expression levels were quantified by real-time RT-PCR, relative to the expression level of β-actin, using the following forward (F) and reverse primers (R) and probes (P), labeled with fluorescent dye (FAM) and quencher (TAMRA). β-actin: F,5′-AGAGGGAAATCGTGCGTGAC-3′; R,5′-CAATAGTGATGACCTGGCCGT-3′; P,5′-FAMCACTGCCGCATCCTCTTCCTCCCTAMRA-3′; next Flk1: F,5′-ACTGCAGTGATTGCCATGTTCT-3′ ; R,5′-TCATTGGCCCGCTTAACG-3′; P,5′-FAMTGGCTCCTTCTTGTCATTGTCCTACGGATAMRA-3′; Vegf: F,5′-AGTCCCATGAAGTGATCAAGTTCA-3′; R,5′-ATCCGCATGATCTGCATGG-3′; P,5′-FAMTGCCCACGTCAGAGAGCAACATCACTAMRA-3′. Reference numbers for primer sequences for mShh and mNetrin-1 are Mm00436528_m1 and Mm00500896_m1, respectively (Applied Biosystems). The percentage of the area occupied by precrossing commissural axons to the total spinal cord area was quantified based on a previously described method (Charron et al., 2003). Briefly, precrossing commissural axon area and total spinal cord area were measured on E11.