Mca409s

The following antibodies were obtained commercially: HCN2 (1:1000; catalog #APC-030, Alomone Labs); CNPase (1:2000; catalog #AMAb91072, Atlas); myelin basic protein (MBP; 1:250; catalog #MCA409S, Serotec); Olig2 (1:100; catalog #AB9610, Millipore); CC1 (1:300; catalog #ab16794, Abcam); MAG (1:100; catalog #MAB1567, Sigma-Aldrich); and CASPR (1:100; catalog #ab34151, Abcam). The Sox10 antibody was a gift from M. Wegner (University of Erlangen, Erlangen, Germany (1:5000).

After perfusion with saline, mouse brains were homogenized and solubilized in 60 mM n-octyl-β-d-glucoside-containing lysis buffer, followed by centrifugation to remove debris. After bicinchoninic acid assay measurement of protein content, lysates were processed by SDS-PAGE followed by western blotting. The primary antibodies used were rat anti-MBP (MCA409S, Bio-Rad, Hercules, CA) and mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (AM4300, Thermo Fisher Scientific, Waltham, MA). Secondary antibodies used were horseradish peroxidase–conjugated anti-rabbit, anti-rat, or anti-mouse IgG.

Cortical slices were washed once with 1x PBS before they were fixed with 4% paraformaldehyde (PFA) (Sigma-Aldrich, Cat# 16005, USA) in PBS for 1 h at RT. The slices were subsequently rinsed in 1x PBS and blocked in 3% Horse Serum (Gibco, Cat# 26050088), 2% BSA, (PanReac AppliChem, Cat# A1391, Germany), and 0.5% Triton X-100 in 1x PBS for 2 h at RT. Following blocking, the slices were incubated for 48 h with primary antibodies at 4°C. The slices were then washed 3 times for 30 min with blocking solution and incubated with 4′,6-diamidino-2-phenylindole (DAPI) (1:1,500, Invitrogen, Cat# MP01306, USA) and the appropriate secondary antibodies overnight at 4°C. The slices were washed again 3 times with blocking solution for 30 min and finally mounted on a glass microscope slide using mounting medium containing Mowiol^® 4-88 Reagent (Millipore, Cat# 475904, Germany). The primary antibodies used were as follows: against myelin basic protein (rat monoclonal anti-MBP, MCA409S, BioRad, 1:300, USA), Neurofilament-H (chicken anti-NF200, Abcam, Cat# ab4680 1:10,000, UK). Secondary antibodies were all purchased from Life Technologies (Life Technologies, 1:1,000, USA). Images were obtained using confocal laser scanning microscopy (Leica microsystems, TCS SP8, Germany) and processed using Fiji image processing package.¹

Mice were transcardially perfused with 4% paraformaldehyde. The spinal cords were removed, postfixed overnight, and cut into 100-μm coronal and sagittal sections using a vibratome (Leica Microsysteme, Rueil-Malmaison, France). Immunofluorescent labeling was performed on sections fixed with paraformaldehyde 4%. The following antibody was used: anti-MBP (rat, 1/500; BioRad MCA409S, CA, United States). The sections and cells were incubated with appropriate Alexa-conjugated secondary antibodies and then counterstained with Hoechst 33258 (1/1,000; Sigma-Aldrich, St. Louis, MI, United States).