Anti-CD62E antibody [ENA2] (ab8165)

Application notes

IA: Use at an assay dependent concentration.
IHC-Fr: Use at an assay dependent concentration.
Agg: Use at an assay dependent dilution.

The antibody is extremely useful for staining of E-selectin expressing endothelial cells. It permits staining of in vitro cultured cells as well as frozen sections. For adhesion inhibition studies the F(ab')₂ ENA2 monoclonal antibody preparation is most efficient. Use of F(ab')₂ prevents adhesion of leucocytes via Fc receptors (2).

Instructions for use:
In vitro cultured cells can be fixed with 1% paraformaldehyde, 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 TNFalpha (1 ng/ml), IL-1 (100 U/ml) or LPS (1 ¥ìg/ml) in tissue culture medium at 37¢ªC. As negative control it is advised to use a control murine IgG1 antibody. For inhibition studies care should be taken to incubate endothelial cells before addition of granulocytes or to use higher concentrations of antibody in case the antibody is added simultaneously.
Not yet tested in other applications.
Optimal dilutions/concentrations should be determined by the end user.