Mice were anesthetized and perfused intracardially with PBS and then 4% paraformaldehyde. Brains were dissected out, postfixed with 4% paraformaldehyde at 4°C for 1h and processed for vibratome section (50 μm). Pregnant mice at indicated gestational days were anesthetized and perfused using PBS. Embryos were dissected out and fixed with 4% paraformaldehyde at 4°C overnight and processed for frozen sections (16 μm).
To visualize PNS myelin, freshly extracted mouse sciatic nerves segments (10 mm) were teased on poly-L-lysine coated microscope slides, using fine needles to gently separate the nerve fibers. The preparations were then dried overnight at room temperature, washed in PBS 3×5 min and finally mounted in 50% glycerol/PBS. Immunostaining with antibodies against Olig2 (Millipore), PDFGRα (BD Bioscience, 558774), Tau (Sigma T6402), CC1 (Oncogene Research, OP80) and MBP (Covance, SMI-94R) or Neurofilament (Covance, SMI-31R), which labels a phosphorylated epitope in extensively phosphorylated neurofilament H and, to a lesser extent, with neurofilament M in axons, was performed on tissue or cell cultures using standard protocols along with corresponding fluorescent secondary antibodies (Jackson lab). Topro3 (Molecular Probe) was included to label nuclei. In situ hybridization was carried out as previously described (Yu et al., 2013 (link)).
To visualize PNS myelin, freshly extracted mouse sciatic nerves segments (10 mm) were teased on poly-L-lysine coated microscope slides, using fine needles to gently separate the nerve fibers. The preparations were then dried overnight at room temperature, washed in PBS 3×5 min and finally mounted in 50% glycerol/PBS. Immunostaining with antibodies against Olig2 (Millipore), PDFGRα (BD Bioscience, 558774), Tau (Sigma T6402), CC1 (Oncogene Research, OP80) and MBP (Covance, SMI-94R) or Neurofilament (Covance, SMI-31R), which labels a phosphorylated epitope in extensively phosphorylated neurofilament H and, to a lesser extent, with neurofilament M in axons, was performed on tissue or cell cultures using standard protocols along with corresponding fluorescent secondary antibodies (Jackson lab). Topro3 (Molecular Probe) was included to label nuclei. In situ hybridization was carried out as previously described (Yu et al., 2013 (link)).
