[Trypan blue and alizarin red S sequential staining with subsequent fixation. A modified technic for the study of the...

A sequential corneal endothelium staining technique with trypan blue (TB)--alizarin red S (ARS)--ethanol-trypan blue is described which can be performed in a few minutes. The first step permits the TB exclusion to be determined as a criterion of normal endothelial cell function. The two following steps permit intercellular staining by ARS and nuclear staining by TB. After each staining procedure, fixation can be performed by methanol or ethanol, both with full-thickness corneal preparations or endothelial flat mounts. In flat preparations, the focussing problems and optical artifacts of full-thickness mounts can be avoided. These modifications render simple sequential staining with subsequent fixation a complement and alternative to specular microscopy and to the customary complicated silver staining of corneal endothelium.