Immunohistochemical localization of ATPase in human dysfunctional corneal endothelium

Previous light microscopic autoradiographic results from our laboratory have shown a quantitative decrease in ATPase pump site density in dysfunctional corneal endothelium. In order to develop antibody techniques to correlate these findings with electron microscopic localization of pump sites, three types of corneas with dysfunctional endothelium (Fuchs' endothelial dystrophy, aphakic and pseudophakic bullous keratopathy) and two types of corneas with functional endothelium (age matched eye bank and keratoconus) were obtained at the time of transplant surgery. Corneas were fixed, frozen and cryostat sections were incubated in a rabbit kidney ATPase primary antibody followed by incubation in an HRP-labeled secondary antibody. Functional endothelia showed dense antibody labeling along the lateral cell membranes and there was a substantial reduction in labeling along the lateral membranes of dysfunctional endothelia. Positive tissue controls of rabbit kidney showed HRP reaction product in the convoluted tubules. Control tissue, incubated in either non-immune serum or primary antibody preabsorbed with ATPase, confirmed the specificity by having diminished or no reaction product. These results provide immunohistochemical confirmation of our autoradiographic data showing a quantitative reduction in pump site density on dysfunctional corneal endothelium.