ENA2 reacts with E-selectin CD62-E, previous designated the Endothelial Leucocyte Adhesion Molecule-1 (ELAM-1). The antibody reacts with human endothelial cells activated with TNF-alpha, IL-1 or endotoxin. The antibody was found to react also with cells transfected with the E-selectin gene. The antibody inhibits the adhesion of granulocytes both neutrophilic and eosinophilic.
Frozen sections, Functional studies, Immuno assays, Immuno fluorescence
FS: the antibody can be used for adhesion inhibition studies for which the antibody is most efficient and
prevents adhesion of leucocytes via Fc receptors. (Ref.3)
IF: In vitro cultured cells can be fixed with 1% paraformaldehyde and kept in PBS plus azide before
staining. Tissue sections are advised to be fixed for 10 min in pure acetone and followed by incubation
for 10 min in chloroform. Incubation with a pretested dilution of the antibody is advised to be followed
by a biotin conjugated anti-murine Ig and a further incubation with an enzyme (alkaline phosphatase)
conjugated streptavidin. For selection of the most useful dilution in a given situation a test staining with
cells or tissue known to express the antigen should be performed. To this end either cultured endothelial
cells or a small fresh skin biopsy can be incubated for 4 hours with TNF-alpha (1 ng/ml), IL-1 (100 U/ml)
or LPS (1 μg/ml) in tissue culture medium at 37°C. As negative control it is advised to use a control
murine IgG1 antibody.
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. For functional studies, in vitro dilutions have to be optimized in user’s experimental setting.
Fresh skin biopsy incubated for 4 hours with TNF-alpha (1 ng/ml), IL-1 (100 U/ml) or LPS (1 μg/ml) in tissue culture medium at 37°C.