Rabbit anti tyrp1

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.

Western blot analysis was performed as described^{24 (link)} with the following primary antibodies: polyclonal rabbit anti-TYR antibody (1:1000, Santa Cruz Biotechnology), polyclonal rabbit anti-β-actin antibody (1:800, Sigma, Germany), polyclonal rabbit anti-K10 antibody (1:1000, Abcam), rabbit anti-TYRP1 (1:1000, Abcam), mouse anti-MITF (1:1000, Thermo, USA), rabbit anti-PAX3 (1:200, Bioss), rabbit anti-MC1R (1:700, Abcam), polyclonal rabbit anti-Cdk5 antibody (1:200, Abcam), rabbit anti-gp100 (1:500, Abcam) and polyclonal rabbit anti-MC4R antibody (1:500, Abcam), rabbit anti-IL1R (1:500, Abcam), rabbit anti-NASM antibody (1:500, Abcam) and rabbit TGFβIIR (1:500, Abcam). Immunoblots were scanned on a ChemiDOC^TM XRS + imager (Bio-Rad, USA), and protein levels were quantified using Image-Pro Plus software (Olympus, Japan).