April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
Protocol for Vital Dye Staining of Corneal Endothelial Cells
Author Affiliations & Notes
  • Sunju Park
    Department of Ophthalmology, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, New York
  • Alan G. Fong
    Department of Ophthalmology, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, New York
  • Hyung Cho
    Department of Ophthalmology, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, New York
  • Cheng Zhang
    Department of Ophthalmology, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, New York
  • David C. Gritz
    Department of Ophthalmology, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, New York
  • Gibran Mian
    Department of Ophthalmology, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, New York
  • Alexandra A. Herzlich
    Department of Ophthalmology, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, New York
  • Patrick Gore
    Lions Eye Bank for Transplant and Research, Tampa, Florida
  • Ashley Morganti
    Lions Eye Bank for Transplant and Research, Tampa, Florida
  • Roy S. Chuck
    Department of Ophthalmology, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, New York
  • Footnotes
    Commercial Relationships  Sunju Park, None; Alan G. Fong, None; Hyung Cho, None; Cheng Zhang, None; David C. Gritz, None; Gibran Mian, None; Alexandra A. Herzlich, None; Patrick Gore, None; Ashley Morganti, None; Roy S. Chuck, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 779. doi:
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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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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract
 
Purpose:
 

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.

 
Methods:
 

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.

 
Results:
 

(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).

 
Conclusions:
 

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.  

 
Keywords: cornea: endothelium • cornea: basic science • pathology techniques 
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