Fluorchem fc2 gel imaging system

Western blotting was used to assess the expression levels of TLR4 and NF-κB after intracerebral hemorrhage. Corpora striata were collected, prepared in lysis buffer, and centrifuged at 13,000 ×g for 5min. Protein concentrations from supernatant were detected using a BCA kit (Beyotime, Haimen, Jiangsu, China). Protein solution, weighing 30mg, was separated by polyacrylamide gel electrophoresis with different concentrations. The gel was then transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA), blocked for 1h in a 5% solution of skim milk (dissolved in Tris-buffered saline plus 0.1% Tween-20 (TBST)). The membranes were incubated with primary antibodies and monoclonal rabbit anti-TLR4 and anti-NF-κB antibody (all diluted 1 : 1000; Abcam, HK, China), respectively, at 4°C overnight. The membranes were washed with TBST 3×10 min on the second day and then were incubated with the secondary antibody conjugated with horseradish-peroxidase (Beyotime). The targeted antigens were detected by standard chemical luminescence methods (Beyotime) with Fluor Chem FC2 gel imaging system (Alpha Innotech, Santa Clara, CA, USA). The expression of TLR4 and NF-κB was determined by using the GADPH protein as the internal reference. Western blots were duplicated with three independent sets. Band intensities were measured with Image J software.