Western Blot Analysis of Cerebral Tissue

Western blot analyses were performed as previously described32 (link). Briefly, cerebral tissue samples were collected, homogenized, and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 10% polyacrylamide gels. A BCA Protein Assay Kit (Beyotime) was used to measure protein concentrations with the bicinchoninic acid method. After separation, protein samples were transferred onto Immobilon nitrocellulose membranes. The membranes were blocked with 5% nonfat milk at room temperature for 1 h. The membranes were then incubated with the following primary antibodies overnight at 4°C: rabbit anti-β-actin (1:1,000, rabbit polyclonal, Abcam, ab8227), rabbit anti-Cleaved-Caspase-3 (1:5,000, Abcam, ab214430), rabbit anti-TLR2 (1:1,000, ab213676), and rabbit anti-TLR4 (1:1,000, rabbit polyclonal, Abcam, ab13556). After washing the membranes with Tris buffered saline tween (TBST) three times, horse radish peroxidase (HRP)-conjugated goat anti-rabbit IgG or goat anti-mouse IgG secondary antibodies (1:5,000) were applied, and the membranes were incubated with the secondary antibodies at room temperature for 1.5 h. The protein bands were detected using a Bio-Rad imaging system (Bio-Rad, Hercules, CA, United States of America) and quantified with ImageJ software.