Affi gel heparin beads

E11.5 hindbrain explants were cultured as described (Tillo et al., 2014 (link)). Affi-Gel heparin beads (Bio-Rad) were soaked overnight in 100 ng/ml VEGF₁₆₅ (PreproTech) or 10 ng/ml FGF2 (Sigma) before implantation. In some experiments, explants were treated with 8.25 mU/ml heparitinase from Flavobacterium heparinum (Seikagaku-Kogyo) in PBS. FBM neuron migration was measured with ImageJ (NIH, Bethesda) as the distance from r5 to the leading group of cells in r6 and normalised to the unimplanted side of each hindbrain. E12.5 trigeminal ganglia were cultured on coverslips coated in poly-L-ornithine and mouse laminin (Sigma) in Neurobasal media containing N2 and B27 (Thermo Fisher) and 4% methylcellulose for 40 h and then fixed in 4% formaldehyde for immunostaining with TUJ1 (Graef et al., 2003 (link)). In some experiments, explants were treated with 200 ng/ml recombinant SLIT1 and/or SLIT2 (R&D Systems) at 2, 18 and 24 h. Quantification of neurite extension was performed with the NeuriteJ plugin of ImageJ.

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.