Optimization of immunostaining on flat-mounted human corneas

The corneal endothelium, a monolayer of tightly packed hexagonal flat cells at the posterior surface of the cornea, is crucial for maintaining corneal transparency. Possibilities for analyzing the protein expressed in corneal endothelial cells (ECs) are limited by their small number (300,000 to 500,000 per cornea). Protein extraction for immunoblots is not the current technique and has been reported only after several donors ’ ECs are pooled [1-4]. Immunostaining techniques are therefore those most widely used to study protein expression in situ. In the literature, the robust technique of immunohistochemistry (IHC) on cross sections is the main technique used for the corneal endothelium because IHC is simple, despite allowing visualization of only a few ECs, without clear subcellular localization, and being subject to the staining artifacts frequently encountered at tissue borders. To avoid these drawbacks, we previously developed specific immunostaining protocols performed directly on the intact endothelium of the whole cornea to allow en face viewing [5] without a tissue cross section. This technique has two main advantages: striking visualization of the whole intact endo-thelium, allowing detection of regional or local difference