Rabbit monoclonal anti beta 3 tubulin antibody

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)).

6 well-plate parallel nanofibers (700nm width, Sigma, Z759333-1EA) were coated with 40 μg/ml of specified protein (FC, Lphn1(Lec-Olf) and its mutant versions) and 100 μg/ml poly-D-lysine (Sigma) in PBS overnight (37°C, 65% humidity and 5% CO2). Next day, plates were washed with PBS and coated with 20 μg/ml laminin in PBS overnight. The next day, plates were washed with PBS and 36 cortical explants (E15.5) were placed per well (6x6 grid, see Figure S5E) and cultured for 2 days in Neurobasal medium supplemented with B27 (Invitrogen) supplemented with 0.4% methyl-cellulose (Sigma). Then the explants were fixed with 4% PFA for 20min, washed with PBS and incubated with rabbit monoclonal anti-beta-III tubulin antibody (Sigma) after 20 min of 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. Mosaic images of each well were taken with SP8 (Leica) microscope. Analysis of the distance covered by cells labeled with DAPI and axons labeled with beta-III-tubulin was done in a semi-automatic mode using a custom ImageJ macro.