The monoclonal antibody 1G11B1 recognizes vascular cell adhesion molecule-1 (VCAM-1). VCAM-1 is a member of the immunoglobulin superfamily of adhesion molecules, which includes ICAMs, PECAM-s and MADCAM, and is involved in leukocyte-endothelial cell interactions. The immunoglobulin superfamily is a type I transmembrane protein characterized by extracellular immunoglobin domains, a transmembrane region and a cytoplasmic tail. They are essential for the development of the embryo and for immune and inflammatory responses. These transmembrane glycoproteins mediate cell interaction with, and adhesion to, other cells and the extracellular matrix. VCAM-1 contains six immunoglobulin domains of the H-type and interacts with VLA-4 expressed on leukocytes. Multiple adhesion molecules play a role in leukocyte recruitment. The process of migration of a leukocyte through the vascular endothelium consists of the following steps: leukocyte-endothelium interaction (first tethering and rolling and than adhesion) and transendothelial migration. VCAM-1 is almost not expressed under physiological conditions. However, under appropriate pro-inflammatory conditions where the endothelium is exposed to inflammatory cytokines such as tumour necrosis factor-α or IL-1b and becomes activated, VCAM-1 gene expression is rapid elevated by the vascular endothelium. There is also a soluble form of VCAM-1 which is angiogenic and chemotactic for endothelial cells. sVCAM-1 is up-regulated in several disease states (eg, myocardial infarction, type 2 diabetes mellitus, primary antiphospholipid syndrome, and rheumatoid arthritis).
Flow cytometry, Frozen sections, Functional studies, Immuno fluorescence, Immuno precipitation
F: Cryostatic sections were fixed in acetone, and incubated with mAb at 10 µg/ml in PBS/0.1% tween20
FC: 1 µg mAb per 1x105 cells
FS: Stimulated EC were preincubated for 30' at 37 °C with mAb and than used in adhesion assay with T-cells
IP: precleared EC lysates were immunoprecipitated with mAB and analyzed by 7.5% SDS-PAGE under reducing conditions
For immunohistochemistry, dilutions to be used depend on detection system applied. It is recommended that users test the reagent and determine their own optimal dilutions. The typical starting working dilution is 1:50.
Activated endothelial cells