Quantification of Demyelinated Protein Levels

Detailed methods are described in our previous studies (Zhan et al., 2008 (link), 2014 (link), 2015 (link)). Briefly, frozen tissues were homogenized in ice-cold RIPA buffer containing a complete protease inhibitor (Sigma). Homogenates were centrifuged at 14,000 × g for 30 min at 4°C. Protein (12.5 μg each) from the supernatant was loaded on 7.5% sodium dodecyl sulfate (SDS) polyacrylamide gels and transferred to the nitrocellulose membrane. The primary antibody included rabbit polyclonal against dMBP (AB5864, 1:1,000 dilutions; Millipore). NIH Image J software was used to quantify band intensities. A mouse monoclonal against β-actin (sc-69879, Santa Cruz, Dallas, TX, USA) was used as a loading control for Western blots and optical densities of each target protein normalized to β-actin. Horseradish peroxidase (HRP) conjugated anti-mouse or anti-rabbit IgG (Bio-rad) was used to detect the primary antibody. The ECL chemiluminescent detection system (PIERCE Inc., Thermofisher Scientific, Waltham, MA, USA) was used to detect the signals. Blots were imaged on the Fluorchem 8900 system (Alpha Innotech, San Leandro, CA, USA). The ratio of the intensity of dMBP/β-actin bands was quantified with NIH Image J software. The relative band intensity in AD samples was averaged and compared to the averaged band intensity of control samples.