Alizarin red staining

A solution of Alizarin red (0.1%) was diluted with 0.9% saline. The deep-iodine coloured solution was filtered (2-μm filter) to remove any undissolved sediment. The pH of the solution was then adjusted to 4.2 with diluted ammonium hydroxide (0.1% solution in normal saline). Excised corneas were placed in plastic vessels with the endothelial side up, immersed in an Alizarin red solution for 90 seconds, and then rinsed three times with saline to wash out the staining reagent. After the staining procedure, corneas were immersed in a 4% paraformaldehyde solution for 10 minutes at room temperature (RT) and then again rinsed three times with saline. Four radial incisions were made across each cornea to flat-mount it endothelial side up and examined under a Nikon Eclipse 50i light microscope (Nikon, Japan). The central cornea was regarded as the region in which the tissue diameter was less than 3mm, whereas the remaining regions were referred to as the peripheral cornea. Three images each were separately captured from the central and peripheral regions of the corneal endothelium in the 4 different quadrants. The ultimate ECDs for the central and peripheral areas were calculated by averaging the 12 ECDs.