Rabbit anti mouse s100 polyclonal antibody

Spinal cord segments L_4–6 obtained 1, 2, 4, and 8 weeks after curcumin administration were fixed for 3 days in 10% neutral formaldehyde before being used for immunohistochemical staining of S100. Briefly, L_4–6 spinal cord slices were placed in 3% H₂O₂ for 10 minutes to block endogenous peroxidase, and then boiled for 10 minutes in 0.01 M sodium citrate buffer (pH 6.0). Slices were then blocked for 15 minutes with anti-species serum and washed with 0.01 M phosphate-buffered saline with Tween 20 (pH 7.4). Rabbit anti-mouse S100 polyclonal antibody (1:500; Beyotime Institute of Biotechnology, Najing, Jiangsu Province, China) was added and incubated at 4°C overnight. Biotinylated goat anti-rabbit IgG (1:1,000; Boster, Wuhan, Hubei Province, China) was added for 20 minutes at 37°C. Streptavidin and biotinylated horseradish peroxidase were added for 20 minutes at 37°C. The labeled antigens were visualized using 3,3′-diaminobenzidine (Jiancheng Bioengineering Institute, Nanjing, Jiangsu Province, China). Brown staining indicated positive protein labeling. Microscopic examination (PM-10A0, Olympus, Beijing, China) was conducted by two pathologists who were blinded to treatments and used Image-Pro Plus 6.0 software (Media Cybernetics Inc.) to analyze the integrated optical density by scanning the stained area.

At 1, 2, 4, and 8 weeks following curcumin administration, L_4–6 spinal cord samples were lysed in ice-cold radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with 12% gels and electroblotted onto polyvinylidene fluoride membranes. The membranes were incubated in rabbit anti-mouse S100 polyclonal antibody (diluted 1:5,000 with PBS containing 1% bovine serum albumin; Beyotime Institute of Biotechnology) at 4°C overnight. They were then incubated with horseradish peroxidase-labeled goat anti-rabbit IgG for 1 hour at 37°C and visualized with 3,3′-diaminobenzidine using a western blotting 3,3′-diaminobenzidine kit (Beyotime Institute of Biotechnology). X-ray film was exposed, scanned, and analyzed. Protein levels were represented as the ratio of the relative grayscale value for the protein of interest to that of GAPDH, and analyzed using Image-Pro Plus 6.0 software (Media Cybernetics Inc.).