Smi 94r

Cells were lysed in lysis buffer (50 mM HEPES-NaOH, pH 7.5, 20 mM MgCl₂, 150 mM NaCl, 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, 1 mg/ml leupeptin, 1 mM EDTA, 1 mM Na₃VO₄, and 10 mM NaF) containing detergents (0.5% NP-40, 1% CHAPS, and 0.1% SDS). These detergents are important for myelin protein isolation [18 (link), 19 (link)]. Unless otherwise indicated, all steps were performed at 4°C. Equal amounts of protein (20 μg total protein) in centrifuged cell supernatants were heat-denatured and prepared for immunoblotting using the TransBlot TurboTransfer System (Bio-Rad). The transferred membranes were blocked with a Blocking One kit and incubated with mouse monoclonal anti-MBP (BioLegend, SMI-94R) and mouse monoclonal anti-CNPase (Sigma-Aldrich, C5922) antibodies, followed by a peroxidase-conjugated secondary antibody. The bound antibodies were detected using a Chemiluminescence ImmunoStar Zeta kit (Wako).

Brains from Tardbp^+/+ and Tardbp^+/Q101X male mice (both n = 3) were harvested for histopathological analysis at one year of age. Mice were perfused transcardially with 0.9% saline followed by 4% paraformaldehyde (PFA). Following dissection, brains were transferred to formalin and subsequently embedded in paraffin wax. Sections were stained with haematoxylin and eosin, and antibodies against GFAP (3 ug/mL, DAKO, Z0334), IBA-1 (1.7 ug/mL, WAKO Chemicals, 019-19741), p62 (5.6 ug/mL, Progen Biotechnik, GP62-C), MBP (2 ug/mL, Covance, SMI-94R) and neurofilament (0.5 ug/mL, Sigma, N5389) using a Ventana automated immunohistochemical staining machine (Ventana Medical Systems, Tuscon, USA). Once incubated with appropriate secondary antibodies, immunoreactivity was developed with 3,30-diaminobenzidine and counterstained with haematoxylin.