title: Protocol for vital dye staining of corneal endothelial cells.
abstract: OBJECTIVE
  To describe a step-by-step methodology to establish a reproducible staining protocol for the evaluation of human corneal endothelial cells.
  
  
  METHODS
  Four procedures were performed to determine the best protocol. (1) To determine the optimal trypan blue staining method, goat corneas were 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, goat corneas were stained with 2 dilutions of alizarin red (1% and 0.5%) and 0.2% trypan blue. (3) To ensure that trypan blue truly stains damaged cells, goat corneas were exposed to either 3% hydrogen peroxide or to balanced salt solution, and then stained with 0.2% trypan blue and 0.5% alizarin red. (4) Finally, fresh human corneal buttons were examined; 1 group was stained with 0.2% trypan blue and another group with 0.4% trypan blue.
  
  
  RESULTS
  For the 4 procedures performed, the results are as follows: (1) trypan blue staining was not observed in any of the normal corneal samples; (2) 0.5% alizarin red demonstrated sharper cell borders than 1% alizarin red; (3) positive trypan blue staining was observed in the hydrogen peroxide exposed tissue in damaged areas; (4) 0.4% trypan blue showed more distinct positive staining than 0.2% trypan blue.
  
  
  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.
authors.affiliation: Department of Ophthalmology and Visual Sciences, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, NY 10467, USA.
authors.forename: Sunju
authors.initials: S
authors.lastname: Park
authors.forename: Alan G
authors.initials: AG
authors.lastname: Fong
authors.forename: Hyung
authors.initials: H
authors.lastname: Cho
authors.forename: Cheng
authors.initials: C
authors.lastname: Zhang
authors.forename: David C
authors.initials: DC
authors.lastname: Gritz
authors.forename: Gibran
authors.initials: G
authors.lastname: Mian
authors.forename: Alexandra A
authors.initials: AA
authors.lastname: Herzlich
authors.forename: Patrick
authors.initials: P
authors.lastname: Gore
authors.forename: Ashley
authors.initials: A
authors.lastname: Morganti
authors.forename: Roy S
authors.initials: RS
authors.lastname: Chuck
mesh_terms: D000818:Animals; D000880:Anthraquinones / Q000737:chemistry*; D004396:Coloring Agents / Q000737:chemistry*; D004728:Endothelium, Corneal / Q000166:cytology* / Q000187:drug effects; D006041:Goats; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D013194:Staining and Labeling / Q000379:methods*; D014343:Trypan Blue / Q000737:chemistry*
chemical_list: D000880:Anthraquinones; D004396:Coloring Agents; C010078:alizarin; D006861:Hydrogen Peroxide; D014343:Trypan Blue