E-selectin, Human, mAb ENA2

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.