Ab224368

Proteins were extracted from the pericontusive cortex (width, 3 mm; depth, 3 mm; length, 5 mm) and quantified using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Western blotting was performed to quantify the expression of OX1R, TLR4, NF-p65, NLRP3, ASC, and Caspase-1. Bicinchoninic acid (Cat. no. PA115; Tiangen Biotech, China) was used to measure protein concentrations in the tissue lysates. The primary antibodies used for western blotting included anti-OX1R (1:1000, ab224368, Abcam, UK), anti-TLR4 (1:1000, ab13556, Abcam), anti-p65 (1:1000, D14E12, Cell Signaling Technology, USA), anti-NLRP3 (1:500, ERP23094-1, Abcam), anti-caspase-1 (1:500, ab207802, Abcam), anti-ASC (1:1000, ab283684, Abcam), anti-GAPDH (1:2000, 60004-1-lg, Proteintech, China), and anti-HDAC1 (1:2000, ab109411, Abcam). Subsequently, proteins were electrophoretically separated on sodium dodecyl sulfate-polyacrylamide gels and transferred on to polyvinylidene fluoride membranes. Secondary antibodies (1:5000, Zsgb-Bio, China) conjugated to horseradish peroxidase were incubated with the membranes for 60 min at ambient temperature to facilitate primary antibody binding. After western blotting, the Image Lab Software (version 3.0) was used to evaluate the results.