Spinal cords from E12.5 BIBW2992 embryos were prepared in an open book configuration. Dissected tissues were lyzed for 30 min at 4°C. Samples were analyzed by western blot, using anti-Plexin-A1
(1/1,000, AbCAM) and anti-β-actin (1/1,000, Sigma) antibodies, as described in Nawabi et al. (2010). For dot blot, the samples were spotted on a nitrocellulose membrane and probed with anti-gdnf antibody (1/500, R&D). FPs were isolated from E12.5 embryos and cultured in three-dimensional plasma clots in Neurobasal medium (GIBCO). The supernatant was collected after 48 hr. For producing regular FPcm, four isolated FPs were grown in 500 μl of medium. For production of the FPcm from the gdnf mouse line, due to the limitations of obtaining more than one homozygote per littermate, one FP was placed in 250 μl of LBH589 medium and was thus diluted by 2-fold, compared with the regular FPcm. The FPs from the different gdnf genotypes were collected from the same littermates and produced concomitantly and diluted the same way. For collapse assay, dorsal spinal cord tissues from E12.5 embryos were dissociated and cells were plated onto polylysin- and laminin-coated glass coverslips
in Neurobasal supplemented with B27, Glutamine (GIBCO), and Netrin-1 (R&D) medium. After 1 or 2 days in vitro, neurons were incubated with control or FPcm or different molecules for 1 hr at 37°C. Then Sema3B-AP was added to cultures for 30 min at 37°C. Cells were fixed in paraformaldehyde (PFA) 4% in PBS/1.5% sucrose and labeled with phalloidin-TRITC (1/500, Sigma). Collapsed growth cones were scored as in
Falk et al. (2005). Statistical comparisons were done with ANOVA-1 test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. about Results from three to ten independent experiments with at least 80 cones per experiment were pooled for the analysis. For coculture experiments, HEK293T cells were transfected with plasmids encoding either Sema3B-Alcaline Phosphatase fusion protein gdnf or control Alcaline Phosphatase. Cell aggregates were cocultured with dorsal spinal cord tissues cut into explants in Neurobasal medium (GIBCO) supplemented with B27 (GIBCO), glutamine (GIBCO), and Netrin-1 (R&D) medium. GDNF (400 ng; Sigma) was added twice to the culture medium. Cocultures and spinal cord explants were grown for 48 hr, fixed in 4% PFA, and stained with anti-Nf160kD antibody. As described in Falk et al. (2005), a qualitative guidance index was attributed to the cocultures to assess the degree of repulsive (negative values) or attractive (positive values) effects.