title: Application of immunogold labelling for light and electron microscopic localization of endothelial leukocyte adhesion molecule 1 (ELAM-1) on cultured human endothelial cells.
abstract: This study describes the expression characteristics of E-selectin molecules using immunogold histochemical techniques on cultured human umbilical vein endothelial cells (HUVEC). The expression of E-selectin was induced by tumour necrosis factor-alpha (TNF-alpha, 300 U/ml), phorbol ester (PMA, 10 ng/ml) and bacterial lipopolysaccharide (LPS, 4 micrograms/ml). No expression was demonstrated on control cells. Using the silver-enhanced colloidal gold-labelling technique, at the light microscopical level, HUVEC could be distinctively subdivided into three staining types. The cell labelling index, expressed as the number of 'positively' stained cells as a proportion of all viewed cells was the highest in the LPS group. For transmission electron microscopy (TEM) the preembedding immunocytochemical staining method and embedding in epoxy resin (Agar 100) according to standard procedures was used. In TEM gold particles were localized in close association with the apical plasma membrane, as well as on the surface of microvillus-like projections (the latter by TNF-alpha group). For high resolution scanning electron microscopy (HR-SEM) the secondary (SEI) and the backscattered electron imaging (BEI) modes were used. Gold particles were randomly distributed over the whole cell surface, although they appeared to be denser in the perinuclear zone. The quantitative evaluation on SE and BE viewing (the number of gold particles per cell area in microns 2) demonstrated the highest density of labelling in the LPS-treated group, but there was only a significant difference between LPS and TNF-alpha groups (P < 0.01, t-test). Furthermore, the ultrastructural studies indicated that treatment with substances which up-regulate E-selectin expression was not related to toxic cell damage or significant alterations of cellular ultrastructure.
authors.affiliation: Institute of Pathology, Johannes Gutenberg University, Mainz, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Tomczok
authors.forename: W
authors.initials: W
authors.lastname: Sliwa-Tomczok
authors.forename: C L
authors.initials: CL
authors.lastname: Klein
authors.forename: F
authors.initials: F
authors.lastname: Bittinger
authors.forename: C J
authors.initials: CJ
authors.lastname: Kirkpatrick
mesh_terms: D015815:Cell Adhesion Molecules / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D002478:Cells, Cultured; D019040:E-Selectin; D004730:Endothelium, Vascular / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D005786:Gene Expression Regulation / Q000187:drug effects; D017979:Gold Colloid; D006801:Humans; D007150:Immunohistochemistry; D008070:Lipopolysaccharides / Q000494:pharmacology; D008853:Microscopy; D008855:Microscopy, Electron, Scanning; D016253:Microscopy, Immunoelectron; D011994:Recombinant Proteins / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014471:Umbilical Veins / Q000166:cytology
chemical_list: D015815:Cell Adhesion Molecules; D019040:E-Selectin; D017979:Gold Colloid; D008070:Lipopolysaccharides; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D013755:Tetradecanoylphorbol Acetate