Mouse monoclonal to rage

For fluorescence staining, neurospheres, adhered to poly-L-ornithine (PLO) pre-coated coverslips, were fixed using 4% paraformaldehyde in 0.01 M phosphate-buffered saline (PBS, pH 7.4) for 30 minutes at room temperature and blocked with 5% v/v fetal bovine serum or with 0.3% v/v Triton-X 100 (Sigma-Aldrich, St. Louis, MO) in PBS. Neurospheres were incubated in mouse monoclonal to RAGE (1:200, Abcam, Cambridge, UK), goat polyclonal to Nestin (1:100, Santa Cruz Biotechnology, CA, USA), or mouse monoclonal to Tubulin (1:100, Beyotime, Beijing, China) overnight at 4 °C and then relative fluorescence secondary antibodies were incubated for 2 hours at room temperature. Cell nuclei were stained with 4′−6-Diamidino-2-phenylindole (DAPI, Beyotime, Beijing, China) for 10 minutes at room temperature. Samples were mounted onto glass slides, and images were captured using a confocal microscope (Carl Zeiss, LSM780, Weimar, Germany) and examined by Zen 2011 software (Carl Zeiss, Weimar, Germany).

Neurospheres in vehicle or 1 ng/ml HMGB1 were homogenized with RIPA (Sigma-Aldrich, St. Louis, MO) supplemented with protease inhibitor cocktail (Roche, Indianapolia, IN, USA). The protein concentration was measured using BCA Protein Assay Kit (Beyotime, Beijing, China). Proteins (20 µg/lane) were separated by 10% SDS-PAGE and electroblotted to polyvinylidene difluoride membranes (Roche, Indianapolia, IN, USA). After membranes were blocked in 5% skimmed milk in TBST at room temperature for 2 hours. Then, they were incubated with mouse monoclonal to RAGE (1:1000, Abcam, Cambridge, UK) and mouse monoclonal to GAPDH (1:1000, Zsgb-bio, Bejing, China) overnight at 4 °C. After incubation with peroxidase-conjugated (HRP)-conjugated secondary IgG (Zsgb-bio, 1:5000) for 2 hours at room temprature. All membranes were detected by ChemiDoc™ XRS⁺ imaging system (Bio-Rad, California, USA) using the WesternBright ECL Kits (Advansta, Menlo Park, CA, USA). Densitometric measurement of each membrane was performed using Image Lab™ software (Bio-Rad, California, USA). GAPDH, an internal control, was used to normalize the expression level of each protein.