Mouse monoclonal anti icam 1

Cells were lysed in boiled Sample Buffer 1x (50 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 100 mM DTT). In total, 30 μg of total protein extracts were resolved on 8% SDS-PAGE and electrically transferred onto poly(vinylidene difluoride) membranes (PVDF, Bio-Rad Laboratories, Hercules, CA, USA) membranes. Membranes were blocked with TBS-T (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.02% Tween-20) containing 5% skimmed milk (Bio-Rad), for 1 h at room temperature, and then incubated overnight at 4°C with primary antibodies diluted in TBS-T containing 5% milk. The following antibodies were used: mouse monoclonal anti-ICAM-1 (Santa Cruz Biotechnology, Dallas, TX, USA; working dilution 1:500), rabbit polyclonal anti-CD36 (Santa Cruz Biotechnology, working dilution 1:500), mouse monoclonal anti-α-tubulin (Sigma-Aldrich, working dilution 1:10,000). After extensive washing, immunecomplexes were detected with horse-radish peroxidase conjugated species-specific secondary antibodies (Jackson Laboratory, Bar Harbor, ME, USA) followed by enhanced chemiluminescence reaction (Millipore Corporation, Billerica, MA, USA). Proteins detected by immunoblotting were quantified by densitometry (ChemiDoc imaging system, BioRad) and normalized as a function of α-tubulin with Image-Lab 5.0 software (Bio-Rad).

After stimulation, HUVECs were fixed in 4% paraformaldehyde and permeabilized in 0.1% Triton X-100/PBS for 5 min. After being blocked with BSA for 30 min, cells were incubated with the primary antibodies rabbit monoclonal anti-HMGB1 (1:1000, Abcam) and mouse monoclonal anti-ICAM-1 (1:500; Santa Cruz Biotechnology) overnight at 4°C. After being washed with TBS-T, cells were incubated with secondary antibodies (1:500; Jackson immunoresearch). A drop of Prolong Gold antifade reagent with DAPI (Vector Laboratories, CA, USA) was used to seal the coverslip. Images were acquired by laser scanning confocal microscopy (LSM 710; Carl Zeiss, Germany). Data were analyzed by use of Image-Pro Plus 6.0.