Anti icam 1 antibody

Western blotting, immunoblotting, and densitometry were performed employing standard protocols [14, 15] . We used the Biorad ChemiDoc XRT+ device. The number of investigated samples/per group was n=4.
Equal amounts of 20 µL lysate, containing 3 µg/µL protein, were loaded onto precast TGX stain-free gels (Bio-Rad, Munich, Germany). Each western blot was performed 3 times. Anti-beta-tubulin, and antivimentin, were used at a dilution of 1:1000 (Cell Signaling Technology, Inc.); anti-osteopontin antibody was used at a dilution of 1:1000 (Rockland Immunochemicals Inc., Limerick, PA, USA) as well as the anti-integrin-beta1 antibody (Epitomics, Burlingame, CA, USA). The anti-SOX9 antibody was applied at a dilution of 1:500 (Life Technologies). In addition, we used an anti-caveolin-1 antibody (1:1000; Abcam).
The anti-ICAM-1 antibody was purchased from Santa-Cruz Biotechnologies (Heidelberg, Germany) and diluted 1: 500. The secondary horseradish peroxidase-linked antibody was used at a dilution of 1:4000 (Cell Signaling Technology, Inc.).
Ponceau S red staining was used as an alternative to housekeeping proteins as loading controls. The membranes were analyzed using ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA; http://rsb.info.nih.gov/ij/), for densitometric quantification of the bands. Ponceau S was evaluated according to standard protocols.

Immunofluorescent imaging was performed under a confocal laser-scanning microscope (TCS SP5, Leica Microsystems, Germany). Briefly, HPAECs were rinsed with PBS before fixation with 4% paraformaldehyde. The HPAECs were then incubated with the anti-p65 NF-κB antibody (1:50, Cell Signaling Technology) and an anti-ICAM-1 antibody (1:50, Santa Cruz Biotechnology) overnight. HPAECs were subsequently incubated with secondary antibody (anti-rabbit-FITC conjugated, Life Technologies Corporation, USA) at 37 °C for 1 h. Coverslips were mounted onto slides using ProLong antifade mounting reagent (Life Technologies Corporation).