Horseradish peroxidase conjugated anti mouse igg

Western blotting was performed as described previously [7 (link)]. Briefly, protein lysates (2 µg/µl) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the resolved proteins were immunoblotted onto nitrocellulose membranes (Whatman, Dassel, Germany). The residual binding sites on the membranes were incubated with mouse monoclonal anti-Ninjurin-1 antibody (1:1,000 dilution, BD Transduction Laboratories, CA, USA). The membranes were washed three times in Tris-buffered saline containing 0.1% Tween-20 and then incubated with horseradish peroxidase-conjugated anti-mouse IgG (Vector Laboratories, Burlingame, CA, USA). Immunoreactive bands were visualized using a BS ECL Plus kit (Biosesang, Seongnam, Korea) according to the manufacturer's protocol. After imaging, the membrane was stripped and reprobed using mouse anti-β-actin antibody (Sigma-Aldrich, St. Louis, MO, USA). Immunoblot signals were detected by Fusion Solo 6X (Vilber Lourmat, Collegien, France) and analyzed using FUSION® software. The expression values were normalized to that of β-actin. The cerebrum band in each panel was used as a standard for normalization, and the relative density of each band was calculated. The results are presented as the mean±standard error.