Anti-E-selectin, Human, mAb ENA1

For immunohistology 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 1:10. 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 IgGantibody.