Anti hfc

50 μg ml⁻¹ Lphn3^Lec–Olf was mixed with 120 μg ml⁻¹ Cy3-conjugated anti-Fc (Life Technologies A11014) in PBS. Matrices (90 μm width)53 (link) were placed on 60-mm dishes and proteins injected. After 30 min incubation at 37 °C, dishes were washed with PBS and matrices removed. Dishes were coated with 50 μg ml⁻¹ Fc protein mixed with 120 μg ml⁻¹ anti-hFc (Jackson ImmunoResearch 109-005-098) for 30 min at 37 °C and washed with PBS. HeLa cells transfected with Unc5D-ires-GFP and/or FLAG-FLRT2 in pHLSec were cultured on the stripes for between 4 and 7 h, before fixing with 2% sucrose/4% paraformaldehyde in PBS for 10–20 min at room temperature. Wild-type and mutant FLRT2 constructs were expressed in HeLa cells on their own or together with Unc5D-ires-GFP, and stained using anti-FLAG (rabbit, Sigma F7425) and Alexa647-conjugated anti-rabbit (Life Technologies A21245) to ensure FLRT2 expression at the cell surface is not affected by the presence of Unc5D. Analysis was performed using ImageJ.

50 μg/ml His-tagged protein was mixed with 120 μg/ml Cy3-conjugated αFc (Life Technologies A11014) in PBS. Matrices (90 μm width) (Knöll et al., 2007 (link)) were placed on 60 mm dishes and proteins injected. After 30 min incubation at 37°C, dishes were washed with PBS and matrices removed. Dishes were coated with 50 μg/ml Fc protein mixed with 120 μg/ml anti-hFc (Jackson ImmunoResearch 109-005-098) for 30 min at 37°C and washed with PBS. Stripes were further coated with 20 μg/ml Laminin in PBS for at least 2 hours and washed with PBS. Cortical neurons (E15.5) or explants (E15.5) were cultured on the stripes in Neurobasal medium supplemented with B27 (Invitrogen) and, in case of explants, 0.4% methyl-cellulose (Sigma). After 24 (neurons) or 48 (explants) hours and fixed with 4% PFA in PBS for 20 min at room temperature (RT). Neurons and explants were washed and incubated with rabbit monoclonal anti-beta-III tubulin antibody (Sigma) after 20 min permeabilization in 1% BSA, 0.1% Triton X-100/PBS. Cy2 anti-rabbit IgG secondary antibody (Jackson ImmunoResearch, cat#111-225-144) was used to visualize the tubulin signal. Nuclei were counterstained with DAPI before mounting. The numbers of beta-III-tubulin+ or DAPI+ pixels on red or black stripes were quantified using ImageJ (version 1.51p) (Schneider et al., 2012 (link)).