Vegf164

Both flanks of adult anaesthetized mice were shaven. The next day, 100 µl of 1% Evans Blue (Sigma-Aldrich) in sterile saline (wt/vol) was injected intravenously through the lateral tail vein. In some experiments, mice received an intraperitoneal injection of pyrilamine maleate (4 mg/kg body weight in 0.9% saline; Sigma-Aldrich) before Evans Blue injection to inhibit release of endogenous histamine. 30 min after Evans Blue injection, 20 µl of PBS or PBS containing 50 ng VEGF164 (PeproTech), 300 ng SEMA3A (R&D Systems), or 50 ng histamine (Sigma-Aldrich) were injected intradermally each at three sites into the flank skin of anaesthetized mice. After 20 min, mice were culled, the skin was separated from the underlying muscle, and the tissue surrounding the injection sites excised (circled in Fig. 1 A) and dried overnight at 55°C. Evans Blue was extracted by incubation in formamide at 55°C overnight and quantified by spectrophotometry at 620 nm after subtraction of background absorbance at 740 nm. Data from the three sites injected with the same agent (ligand or PBS) were averaged and expressed as fold change relative to PBS control per each mouse. In some experiments, the inner side of the skin was imaged on an MZ16 stereomicroscope (Leica) equipped with a Micropublisher camera (Perkin-Elmer). In other experiments, a sample of liver or skin tissue was retained for immunoblotting.

HDMECs were cultured in MV2 media with supplements (Promocell). MBECs were isolated from mice between 1 and 3 wk of age and cultured on tissue culture plates coated with 20 μg/ml FN in EGM2 media (Lonza) without passaging; 4 µg/ml puromycin (Sigma-Aldrich) was included in the media for 2 d to eliminate contaminating cell types. MLECs were isolated from mice between 1 and 2 mo of age by magnetic-activated cell sorting (MACS) with PECAM1 and ICAM2 antibodies (BD). MLECs were cultured on tissue culture plates coated with 10 µg/ml FN in DMEM-GlutaMAX supplemented with 20% FBS, nonessential amino acids (Life Technologies), and ECGS (Sigma-Aldrich). Cells were stimulated with 50 ng/ml VEGF165 (PeproTech; for HDMEC) or VEGF164 (for MLECs or MBECs) for the indicated times. In some experiments, HDMECs were incubated with inhibitors dissolved in DMSO or the same concentration of DMSO 30 min before VEGF165 stimulation. We used the following inhibitors: 10 µM Imatinib (Cambridge Bioscience), 10 µM PP2 (Sigma-Aldrich), and 0.1 µM PTK/ZK (Vatalanib; Selleckchem).

Hindbrain explants were cultured as previously described (Schwarz et al., 2004 (link); Tillo et al., 2014 (link)). Affi-Gel heparin beads (Bio-Rad) were soaked overnight in 100 ng/ml of VEGF₁₆₄ in PBS (Preprotech) or VEGF₁₈₈ (Reliatech). FBM neuron migration was measured with ImageJ (NIH) as the distance travelled from r5 to the leading group of cells in r6 in each hindbrain and normalised to the control side of each hindbrain.