Anti cd40

Three samples were isolated from each patient: macroscopically normal abdominal aorta, incipient ASVD lesion, and a complicated lesion. Sections were H/E-stained and classified according to the modified classification of American Heart Association (AHA) [6 (link)]. Formalin-fixed, paraffin-embedded aortas, were sliced at 4 μm and stained with polyclonal anti-CD3, anti-S100 (Dako, Glostrup, Denmark), anti-CD40, anti-CD40L (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-NF-κB-p65 (phosphor S536) (Abcam, Cambridge, UK) antibodies. Sections were counterstained with hematoxylin to make nuclei evident. Human parotid glands or ganglions were used as positive controls. Isotype controls were performed using antibody buffer supplemented with irrelevant immunoglobulines of the same isotype, species and concentration as the primary antibody (ThermoFisher, Rockford, IL USA). Negative controls were performed by omitting primary antibodies. Positively stained cells were counted at x100 to x200 and acquired with a digital camera. The percentage of positive cells was assessed irrespective of the staining intensity. Results were expressed as the percentage of each population regarding the total number of cells in the intima or adventitia.

GC (PubChem CID: 161120), Aspirin (PubChem CID: 2244), 2, 3, 5-Triphenyltetrazolium chloride (TTC), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and chloral hydrate were bought from Sigma (St. Louis, MO, United States). DMEM medium (high glucose) and newborn calf serum were products of Gibco (Grand Island, NY, United States). Trypsin was purchased from Amresco (Solon, OH, United States). TNF-α, IL-1β and IL-6 ELISA kits were bought from Sigma (St. Louis, MO, United States). Anti-ICAM-1, anti-VCAM-1, anti-iNOS, anti-CD40, anti-NF-κB p65, anti-p-IκB-α, anti-IKK-β, anti-β-actin, anti-Histone, goat anti-rabbit and anti-mouse IgG antibodies were purchased from Santa Cruz (Santa Cruz, CA, United States). Nuclear-Cytosol Extraction Kit and ECL plus kit was purchased from Jiancheng (Nanjing Jiancheng Bioengineering Inc., Nanjing, China)