Chicken anti p0

Cell cultures were fixed with 4% PFA for 10 minutes at room temperature, and then incubated for 1 hour with 10% normal donkey serum in PBS-T (PBS supplemented with 0.3% Triton X-100 for intracellular antigens). Antibodies used were: mouse anti-NESTIN (1:100; Novus Biologicals, Littleton, CO, USA), rabbit anti-P75^NTR (1:200; Millipore), mouse anti-SOX10 (1:200; R&D system), rabbit anti-S100β (1:200; Dako), rat anti-MBP (1:250 Millipore), chicken anti-P0 (1:100 Millipore), rabbit anti-TYRP1 (1:100; Abcam), rabbit anti-NeuN (1:500; Millipore), rat anti-Neurofilament (1:500; Millipore), rabbit anti-SOX9 (1:500; Millipore) and rabbit anti-TWIST (1:500; Millipore). Cells were incubated with primary antibodies diluted in PBS or PBS-T for 2 hours at room temperature or overnight at 4°C. After three PBS washes, cells were incubated with Rhodamine Red X-conjugated secondary antibody (1:500; Jackson Immunoresearch Laboratories, West Grove, PA, USA) for 1 hour at room temperature. Nuclei were then stained using Hoechst 33342 (Molecular Probes, Life Technologies) and preparations were mounted in Q Path Safemount. The same protocol was used for immunocytochemistry on spheres but before fixation with PFA spheres in suspension were put on slides using cytospin centrifuge (Thermo Scientific) during 5 minutes à 40 rcf.

For western blot analysis and immunohistochemistry the following antibodies were used: goat anti-ChAT (Millipore); rat anti-LAMP1 (Iowa Hybridoma bank); Guinea pig anti-P62 (Progen); rabbit anti-LC3 (Sigma); rat anti-MBP (Chemicon); rabbit anti-NF-H (Millipore); mouse anti-TUJ1 (Promega); mouse anti-tubulin (Sigma); rabbit anti-actin (Sigma); mouse anti-FIG4 (Neuromab); rabbit anti-S100 (Sigma), rabbit anti-NGF receptor p75 (Millipore); chicken anti-P0 (Millipore) and mouse anti-P0 antibodies (kindly provided by J.J. Archelos); rabbit anti-ubiquitin (Santa Cruz); rhodamine phalloidin (Molecular Probes).
Protein lysates were prepared using a lysis buffer containing 1% Triton X-100 (or NP40 1% for autophagy marker evaluation), 50 mm Tris buffer, pH 8.0, 150 mm NaCl, 10 mm NaF, 1 mm Na vanadate, Complete (Roche) protease inhibitors.