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BH Jeng, DM Meisler, JG Hollyfield, KS Aulak, DJ Stuehr; Immunohistochemical Localization of eNOS, iNOS, bNOS, and Peroxynitrite in Stored Human Corneas . Invest. Ophthalmol. Vis. Sci. 2002;43(13):138.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We have previously shown that nitric oxide (NO), the product of nitric oxide synthase (NOS), is continuously released by corneas in storage media. In this study, immunohistochemical techniques were used to localize the 3 isoforms of NOS (eNOS, iNOS, bNOS) and peroxynitrite (a toxic byproduct of nitric oxide and superoxides) in stored human corneas. Methods: Human corneas were stored in Optisol GS media for either 4 or 29 days and were fixed in formalin, embedded in paraffin, and sectioned. Mouse monoclonal antibodies to eNOS, iNOS, bNOS, and nitrotyrosine (to localize peroxynitrite) were used as primary antibodies. Secondary antibody and peroxidase treatment were performed using a biotinylated rabbit anti-mouse antibody horseradish peroxidase system (Enhanced DAB kit, Ventana Medical Systems, Tuscon, Arizona). Sections for negative controls were treated the same except that primary antibody was not used. All sections were counterstained with hematoxylin and photographed with a digital camera system. Results: eNOS was localized to the corneal epithelium in similar quantities at 4 and 29 days of storage. iNOS and bNOS were not localized in corneal tissue. Peroxynitrite was also localized to the epithelium of human corneas, but there was significantly more staining in tissue stored for 29 days compared to tissue stored for 4 days. Conclusion: The presence of eNOS immunoreactivity in stored human corneal tissue corroborates our previous finding that NO is generated by corneas stored in Optisol GS media. The accumulation of peroxynitrite suggests that stored corneal tissue may be subjected to NO-mediated damage.
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