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J. Casiglia, A.R. Ahmed; Laser Scanning Confocal Microscopy Studies of Hemidesmosomal Antigens in Conjunctival Mucosa . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3804.
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Purpose: Samples of human conjunctiva were obtained from cataract surgery on patients without any inflammatory ocular disease. Methods: Samples were immediately fixed, and subsequently embedded in OCT, and sectioned into 8 micron sections. The samples were then placed on slides and stained with primary antibodies to antigens present in hemidesmosomal structures. Primary antibodies were diluted at a concentration of 1:500 and incubated overnight at 4 C. Secondary (flurochrome-conjugated) antibodies were then added, and the process repeated for double or triple-staining as necessary. Antigens evaluated included BP180 (BPAg2), alpha6Beta4 integrin, collagen VII, and laminin 5. Results: Fluorescent staining demonstrated colocalization of subunits of alpha6beta4 integrin, as well as localized staining of these subunits with the BP180 antigen. Collagen VII also stained strongly beneath the basement membrane. Interestingly, laminin 5 could not be visualized—at least not to the same extent as demonstrated in similar studies with normal human oral mucosa and normal human epidermis. Conclusions: This study is the initial step in the use of confocal microscopy to structurally describe the relationships of these critical molecules.
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