Western Blot Quantification Protocol

Western blot analysis was performed as previously described^{15 (link), 17 (link)}. Ipsi- and contralateral grey matter tissues were sampled. Protein concentration was determined by Bio-Rad protein assay kit (Hercules, CA). An equal amount of protein (50 μg) was suspended in loading buffer, denatured at 95°C for 5 minutes and loaded on an SDS-PAGE gel. After being electrophoresed and transferred onto Hybond-C pure nitrocellulose membrane (Amersham), the membrane was blocked with nonfat milk buffer for 1 hour and then incubated with the primary antibodies. The primary antibodies were polyclonal rabbit anti CD163 (Abcam, Ab87099, 1:1000), rabbit monoclonal anti hemoglobin subunit alpha (Abcam, Ab92492, 1:2000), monoclonal mouse anti-GAPDH (Fitzgerald, 10R-G109A, 1:1000000) and monoclonal mouse anti-β actin (Sigma-Aldrich, A3854, 1:100000). The membranes were then incubated with horseradish perixidase-conjugated secondary antibodies for 1 hour at room temperature. The second antibodies were HRP-conjugated goat anti-mouse IgG (1:2000, Bio-Rad) and HRP-conjugated goat anti-rabbit IgG (1:2000, Bio-Rad). Protein band densities were detected by Kodak X-OMAT film and quantified by NIH Image J. CD163 levels were expressed as the ratio of CD163/GAPDH or CD163/β actin. Hemoglobin level was expressed as the ratio of hemoglobin/GAPDH.