The NF-kappa B signal transduction pathway in aortic endothelial cells is primed for activation in regions predisposed to atherosclerotic lesion formation -- Data Supplement - Supplemental Data -- Proceedings of the National Academy of Sciences

Supplementary material for Hajra et al. (2000) Proc. Natl. Acad. Sci. USA 97 (16), 9052-9057.

Detailed Methods
Harvesting of Mouse Aortas.
The arterial tree of halothane-anesthetized mice was perfused via the left ventricle. PBS (pH 7.4) was infused for 5 min, and then 2% paraformaldehyde was infused for 15 min. The perfusion pressure was maintained at 100 mmHg (1 mmHg = 133 Pa) with a pressurized gas cylinder and regulator. Thoracic aortas were harvested, and periaortic adipose tissue was dissected while immersed in cold PBS. Aortas were stored in 2% paraformaldehyde (4ºC) overnight before immunostaining. Silver Nitrate Staining.
Mouse aortas were perfused with PBS (5 ml over 1 min), 0.25% silver nitrate dissolved in distilled water (5 ml over 30 sec), and then PBS again (2.5 ml over 15 sec). Perfusion was via the left ventricle by using 10-ml syringes. Pressure fixation followed, by using 2% paraformaldehyde infused for 15 min at 100 mmHg. Harvested aortas were mounted on the same day and photographed by using a standard light microscope. Immunofluorescence Staining Reagents and Protocols.
Polyclonal and monoclonal primary antibodies were used. Polyclonal antibodies to NF-kB and IkB peptides were purchased from Santa Cruz Biotechnology as purified IgG. They included goat anti-p65 (C-20, 20 C-terminal amino acids) (sc-372-G, 1:500 dilution), goat anti-IkBa (C-21) (sc-371-G, 1:400 dilution), rabbit anti-IkBb (S-20) (sc-946, 1:200 dilution). Phospho-IkBa (Ser32) polyclonal rabbit antibody was purchased from New England Biolabs and used at a 1:20,000 dilution. This antibody reacts with IkBa only when serine 32 is phosphorylated. Other antibodies included monoclonal rat anti-mouse E-selectin IgG2a (PharMingen, 1:500 dilution), monoclonal rat anti-mouse intercellular adhesion molecule (ICAM)-2 IgG2a (PharMingen, 1:200 dilution), monoclonal rat anti-mouse vascular cell adhesion molecule-1 (VCAM-1) (M/K-2.7, American Type Culture Collection, IgG1, undiluted tissue culture supernatant), and polyclonal goat anti-mouse PECAM-1 (M-20, sc-1506, Santa Cruz, 1:200 dilution).

Negative controls included nonimmune goat, rabbit, or rat IgG (Jackson ImmunoResearch; same species and dilution as the primary antibody). For anti-p65, the primary antibody was incubated with blocking peptide-immunogen (sc-372P, Santa Cruz, 1:50 dilution). Secondary antibodies included biotin-conjugated donkey polyclonal anti-goat, -rabbit, or -rat IgG (Jackson) or Cy3-labeled donkey anti-goat or anti-rat.

The immunostaining protocol varied depending on the antibody. Tyramide signal amplification (NEN) was used for detection of p65, IkBa, IkBb, phospho-IkBa, E-selectin, and ICAM-2. Endogenous peroxidase activity was blocked by incubating aortas with 3% hydrogen peroxide in PBS (10 min). Tissues were permeabilized with 0.2% Triton X-100 in PBS (5 min) and blocked with Tris·HCl blocking buffer (NEN, 30 min). For p65, IkBa, IkBb, and ICAM-2 primary antibody incubations were for 1 hr at 22ºC. For E-selectin and phospho-IkBa, primary antibody incubations were overnight at 4ºC. Subsequent steps included biotin-conjugated polyclonal secondary antibodies (30 min), steptavidin-conjugated horseradish peroxidase (30 min), and tyramide complexes conjugated with FITC or Cy3 for IkBb (8 min). All antibodies were diluted in Tris·HCl blocking buffer (NEN) and washes between steps used Tris·HCl washing buffer (0.1 M Tris·HCl, pH 7.5/0.15 M NaCl/0.05% Tween 20). Nuclei were counterstained with propidium iodide (Molecular Probes, 1:1,000 dilution, 30 min) for FITC-tyramide or green nucleic acid stain (Sytox, Molecular Probes, 1:100 dilution, 30 min) for Cy3-tyramide.

Fluorescent-labeled secondary antibodies were used to detect VCAM-1 and PECAM-1 expression. Aortic cells were permeabilized with 0.2% Triton X-100 (5 min) and blocked with 33% donkey serum in PBS (The Jackson Laboratory). The primary antibody to PECAM-1 was incubated for 1 hr at 22ºC, whereas an overnight incubation at 4ºC was used for anti-VCAM-1. The secondary antibodies were Cy3-labeled donkey anti-goat or anti-rat (1:200 dilution, 30 min, 22ºC). Aortas were washed three times with PBS after antibody incubations. Nuclei were counterstained with Sytox.