E-cadherin is a calcium dependent adhesion molecule with important roles in epithelial intercellular adhesion and cellular structure. Immunofluorescent staining can localize and quantify protein expression in cells or tissues. Co-localization of 2 or more different proteins used in combination with fluorochromes of different colors can reveal the location of each protein. We investigated the use of e-cadherin immunofluorescence in different species (rat and mouse), with different fixatives (10% neutral buffered formalin, 4% paraformaldehyde, and 2.5% glutaraldehyde), with and without antigen retrieval, and as a dual label with immunofluorescence for other proteins. Mouse monoclonal anti-e-cadherin antibodies (BD Biosciences, San Jose, CA) in conjunction with fluorochrome-conjugated donkey anti-mouse antibodies clearly delineated sites of e-cadherin expression in lungs of rats or mice. EDTA heat-induced epitope retrieval was required to restore antigenicity to fixed tissues. E-cadherin could be demonstrated in formalin or paraformaldehyde fixed tissues but not in glutaraldehyde fixed tissue. Immunofluorescent double-labeling with e-cadherin can be used with immunofluorescent detection of podoplanin, a lymphatic endothelial cell marker that is also expressed by alveolar type I cells in the lung. Low levels of e-cadherin expression in type I cells allowed the double labeled type I cells to be distinguished from lymphatic endothelium and facilitated diagnosis of lymphangiectasia. E-cadherin immunofluorescent double labeling can also be used with activated caspase 3 or â-catenin immunofluorescence to localize the expression of these proteins in damaged airways. Thus, in the lung, e-cadherin immunofluorescence can demonstrate abnormal and normal epithelial cell junctions, facilitate demonstration of airway epithelial changes, and distinguish podoplaninexpressing alveolar type I cells from lymphatic endothelium.
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