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Sunju Park, Alan G. Fong, Hyung Cho, Cheng Zhang, David C. Gritz, Gibran Mian, Alexandra A. Herzlich, Patrick Gore, Ashley Morganti, Roy S. Chuck; Protocol for Vital Dye Staining of Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):779.
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To date, no clearly delineated staining protocol has been described for assessment of human corneal endothelial cells. We describe a step-by-step methodology to establish a reproducible protocol.
Fresh corneas were removed from 3 goat eyes. (1) To determine the optimal trypan blue staining method, one cornea was divided into quadrants and stained with 4 dilutions of trypan blue (0.4%, 0.2%, 0.1%, and 0.05%) and 1% alizarin red. (2) To determine the optimal alizarin red staining method, a second cornea was divided into halves and stained with two dilutions of alizarin red (0.5% and 1%) and 0.2% trypan blue. (3) To ensure that trypan blue truly stains damaged cells, a third cornea was divided with one half exposed to 3% hydrogen peroxide and the other to balanced salt solution only, then stained with 0.2% trypan blue and 0.5% alizarin red. (4) Finally, 2 fresh human corneal buttons (Lions Eye Institute, Tampa, FL) were examined to refine the protocol for human endothelial cells. One button was stained with 0.2% trypan blue and one with 0.4% trypan blue. Both were stained with 0.5% alizarin red. Tissue samples were flat-mounted and imaged at 100x using the Carl Zeiss Axio Observer inverted digital light microscope.
(1) Trypan blue staining was not observed in any of the samples. (2) 0.5% alizarin red demonstrated sharper cell borders than 1% alizarin red (Figure A). (3) Positive trypan blue staining was observed in the hydrogen peroxide exposed tissue in areas denuded of endothelial cells. (4) 0.4% trypan blue showed more distinct positive staining in damaged areas than 0.2% trypan blue. Figure B shows damaged cells (arrows), exposed Descemet's membrane (arrowheads), and a large denuded area (asterisk).
We were able to determine the optimal vital dye staining conditions for human corneal endothelial cells using 0.4% trypan blue and 0.5% alizarin red. This protocol is reproducible and can be applied with confidence that damaged areas will be correctly identified.
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