Dual staining of corneal endothelium with trypan blue and alizarin red S: importance of pH for the dye-lake reaction

Evaluation of corneal endothelial integrity by combined staining with the vital stain trypan blue and the intercellular stain alizarin red S provides a simple, quick technique for visualisation of both damaged and normal cells, thereby permitting the quantification of endothelial cell damage. Adjustment of the pH of the alizarin red S reagent to 4.2 is important for optimum dye-laking at the intercellular borders, and brief fixation with glutaraldehyde maintains the staining effect of both dyes.