G boxichemi camera

Western blotting was performed in hippocampal homogenates (n = 5 for each time, including single control) as previously described [12 (link)]. After electrotransfer, the membranes were incubated with 5 % skimmed milk powder to block non-specific sites prior followed by washing with TBS-T (0.1 % Tris-buffered saline with 0.05 % Tween 20, pH 7.4). Subsequently, the membranes were incubated with primary antibodies (see Table 6). Then, the membrane were washed with TBS-T and incubated with HRP-labeled anti-mouse (for anti-catalase and anti-β-actin), anti-goat (for anti-IFN-γ) or anti-rabbit (anti-caspase 3, SOD-1, anti-GFAP, anti-NeuN and anti-TNF-α) secondary antibody (1:1000, Sigma Aldrich). Bands were visualized using a chemiluminescence kit (Super Signal West Pico Chemiluminescent Substrate; Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer’s instructions. The luminescent signal from bands was captured by a G:BoxiChemi camera (Syngene, Cambridge, UK) and band intensities were quantified using Image J 1.45S (NIH, Bethesda, MD, USA). Blots were stripped and reprobed for β-actin to monitor protein loading the efficiency of blot transfer, and non-specific changes in protein levels.

After treatment, U87 MG cells were washed with PBS and lysed in ice-cold lysis buffer (25 mM HEPES, 1.5% Triton X-100, 0.1% sodium dodecylsulfate (SDS), 0.5 M NaCl, 5 mM EDTA, and 0.1 mM sodium deoxycholate) containing a protease inhibitor cocktail. Protein concentrations were quantified using a bicinchonic acid protein assay kit (Thermo, San Jose, CA). An equal amount of proteins from each group was separated using SDS-polyacrylamide gel electrophoresis (PAGE), followed by transfer to nitrocellulose membranes. Membranes were incubated with a 5% skim milk solution (blocking solution) for 1 hour, and then incubated with anti-VEGF and anti-β-actin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C for 16 hours. Membranes were probed with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature, and then imaged using a Syngene G:BOX iChemi camera (Syngene, Cambridge, UK) and GeneSnap software (vers. 7.09, Syngene). β-actin was used as an internal control. The density of bands was determined with Gel-Pro Analyzer densitometry software.