Hyperfilm

Spinal cord tissues were collected from deeply anesthetized animals perfusing transcardially with 50 mL of 0.9% NaCl on day 14. Protein concentrations were determined by BCA Protein Assay (Applygen, Beijing, China). Proteins (50 μg) were loaded for each lane and separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis. The proteins were then transferred and incubated overnight at 4 °C with Anti-MCP1 (CCL2) antibody (ab25124) (1:1000, mouse; Abcam, Cambrige, MA, USA.), CCR2 antibody (36784) (mouse, 1:1000, mouse; Signalway Antibody LLC, College Park, MD, USA.); GAPDH antibody (1:10,000, mouse; Sigma-Aldrich Co. LLC., (St. Louis, MO, USA), was used as loading control. These blots were further incubated with horseradish peroxidase–conjugated secondary antibody, developed in enhanced chemiluminescence solution, and exposed on Hyperfilm (Bio-Rad, Hercules, CA, USA,) for 1 to 5 min. Specific bands were evaluated by apparent molecular size. The intensity of the selected bands was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Cellular proteins were extracted from the primary microglial cells and astrocytes using RIPA buffer. The homogenates were centrifuged for 15 min at 12,000× g at 4 °C. The quantity of protein in each supernatant was determined using a BCA Protein Assay kit and then processed as shown above.