Ecl prime western blotting detection reagent

After brains were harvested, protein was extracted. Brains were weighed ( $\sim 300\mathrm{mg}$ ) and suspended in $1\mathrm{mL}$ NPER Reagent buffer (Product #87792, Thermo Scientific) containing protease inhibitors (Roche complete mini protease inhibitor cocktail tablets #04693124001, Sigma). Tissue was then disrupted using a homogenizer on setting 6 (Tissue Tearor #985370, Cole Parmer) and incubated on ice for 10 min. Lysates were cleared by centrifugation at $\mathrm{10,000}\times g$ for 10 min. Protein concentration of the resulting supernatant was measured using Pierce BCA Protein Assay Kit. Brain tissue lysates with total protein of $50\mathrm{\mu }\mathrm{g}$ were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and blocked in 5% nonfat milk for 1 h. Blots were incubated overnight in primary antibody C5, $\mathrm{C}5\mathrm{\alpha }$ , CD88, and TLR4 (Table S2). After washing with TBS-Tween™-20, blots were incubated in appropriate secondary antibodies (Table S2) in 5% nonfat milk in TBS-Tween™-20. Blots were developed with GE Health Care ECL prime western blotting detection reagent and imaged using a CCD camera (G:BOX, Syngene; image resolution 4 megapixels). Density of proteins of interest were normalized to GAPDH (Table S2). The observer was blinded to treatment group.