P75ntr antibody

α-ZAL was a gift from Professor Shunling Dai at Perking Union Medical College. 17β-E2 and 5′-bromo-2′-deoxyuridine (BrdU) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). The protease inhibitor mixture and BCA Protein Assay Kit were purchased from Pierce Biotechnology (Rockford, IL, USA). BrdU antibody was purchased from Millipore (Billerica, MA, USA). BDNF, TrkB, and p75NTR antibody were purchased from Abcam (Abcam, England); β-actin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The avidin-biotin-peroxidase and DAB kits for immunohistochemical detection were obtained from Zhongshan Goldenbridge Biotechnology (Beijing, China). All other chemicals used were of the highest grade commercially available.

MNs were isolated from the spinal cord of CD-1 mice (embryonic day 12–13 fetuses) as previously described^{33 (link),55 (link)}. MNs were harvested in Hank’s balanced salt solution (HBSS, Lonza) containing 1% Toll-like receptor (TLR) trypsin (Worthington) for 15 min at 37 °C, followed by treatment with 1% TLR trypsin inhibitor (Sigma). To collect purified MNs, dissociated spinal cord pieces were incubated in an immunopanning culture dish pretreated with p75^NTR antibody (Abcam) at room temperature for 1 h. The immunopanning culture dish was washed three times with neurobasal medium (Gibco) containing 1 × Glutamax Ι (Gibco) to remove nerve fragments and p75^NTR-negative cells. MNs bound to the bottom of panning dish were treated with depolarization solution (0.8% sodium chloride, 30 mM potassium chloride, and 2 mM calcium chloride, Merck) and gently harvested in coculture medium.