Neogenin

Cells were passaged into 24-well plates containing poly D-lysine (50 μg/ml) coated glass coverslips and after experimental observation were fixed with 4 % paraformaldehyde (PFA), rinsed and incubated overnight in primary antibody. After further rinsing with PBS, cells were incubated in the appropriate secondary fluorescent antibody at 1:200 dilution in PBS with 0.3 % Triton X (1 h) followed by rinsing and mounting the coverslips with Vectashield® fluorescent mounting medium.
Primary antibodies used were: DCC 1:500 (BD Pharmingen, Oxford, UK, #554223); doublecortin (DCX) 1:200 (Santa Cruz, California; #SC8066), neogenin 1:500 (Santa Cruz, California; #SC6536).
Secondary antibodies (all 1:200) were AlexaFluor 594 goat anti-mouse #A11032; AlexaFluor 488 goat anti-rabbit #A11008; AlexaFluor rabbit anti-goat #A11078 (Life Technologies, Paisley, UK).

E18 mouse cortical neurons were plated on poly-L-Lysine (PLL)-coated glass coverslips treated with laminin (Invitrogen; 10 mg/ml) for 24hr and then immunostained with βIII-tubulin (Sigma; 1:500), AE12-1 (1:250), AE12-1Y (1:250) or hIgG (1:250). Embryonic cortical neurons were also stained with F-actin (Molecular Probes; 1:100) and Neogenin (Santa Cruz; 1:200). For the neurite outgrowth assay, E18 mouse cortical neurons were plated on PLL-coated glass coverslips treated with laminin (Invitrogen; 10 mg/ml) and RGMa proteins (5 mg/ml) and incubated for 24hr at 37 °C with control hIgG antibody (1 mg/ml) or anti-RGMa (AE12-1 or AE12-1Y; 1 mg/ml). Cultures were fixed with 4% paraformaldehyde 24hr after plating, and neurons were immunostained with βIII-tubulin (Sigma; 1:500). Experiments were done in duplicates and repeated 3 times. Thus 6 coverslips of neuronal cultures were counted from several areas of the coverslip, and at least 20 neurons were measured per coverslip, as previously described^{14 (link), 16 (link)}. Fiber length was quantified using Image Pro 5.0.