Fitc conjugated anti rage monoclonal antibody

The skeletal muscles from animals at 1 and 2 weeks post ligation were excised, specimens were embedded in TissueTec (Sakura) and snap-frozen in -150 °C methylbutane. Frozen sections (5 µm) were fixed with ice-cold acetone, incubated with FITC-conjugated anti-RAGE monoclonal antibody (1:100, Abcam, USA) for 1 h at room temperature, mounted with DAPI Fluoromount (Southern Biotech, USA), and visualized with a Leica SP8 scanning confocal microscope; images were processed with commercial software (Leica Application Suite X, Leica, Germany). The images were quantified for extent (percentage area) of positive staining in randomly chosen high-powered (200×) fields using algorithms previously validated by our group.

HUVEC cells after the treatment with high glucose concentration were lifted using Accutase (Sigma-Aldrich, USA) and centrifuged for 2 min at 300 ×g at 4 °C. The supernatant was discarded, and the cell pellets were resuspended in FACS buffer (10% FBS, 0.01% sodium azide in PBS, pH 7.4). For the competitive binding study, cells were pre-treated with 1:100 dilution of unlabeled anti-RAGE blocking antibody (R&D systems, USA) for 1 h at 37 °C. Cells were subsequently incubated in various concentrations of Rho-G4-CML (125-750 nM) for 1 h at 37 °C, washed twice, resuspended in FACS buffer and analyzed. Percent binding reduction was found by calculating the percent change of the median Rho-G4-CML fluorescence intensity of anti-RAGE-treated cells compared to control cells. For colocalization studies, cells were incubated with FITC-conjugated anti-RAGE monoclonal antibody (Abcam, USA) for 1 h at 4 °C, washed twice with PBS and incubated with nanoparticles for 1 h at 4 °C. Following the incubation, cells were washed twice with PBS and analyzed by FACS ARIA III (Becton-Dickinson, USA). Vertical and horizontal thresholds were determined using fluorescence minus one (FMO) controls.