All corneal buttons were transferred to 4% paraformaldehyde immediately after collection. The tissue was fixed overnight at 4°C, after which two smaller (2- to 3-mm2) blocks were dissected from the central region of each button and washed with PBS. One of the two blocks was for SHG imaging microscopy and the other for immunofluorescence analysis of αSMA. For the latter, each corneal block was permeabilized by exposure to acetone for 5 minutes at −20°C, washed with PBS, incubated first overnight at 4°C in 50% TD buffer (TD buffer: 137 mM NaCl, 25 mM Tris HCl [pH 7.4], 0.7 mM Na2HPO4, 5 mMKCl) containing 3% BSA and then for 72 hours at 4°C with FITC–conjugated mouse monoclonal antibodies to αSMA (1:100 dilution; Sigma, St. Louis, MO) and Syto59 dye (1:1000; Molecular Probes, Carlsbad, CA) in 50% TD buffer, and finally washed three times for a total of 3 to 4 hours with PBS. Corneal blocks for both SHG imaging microscopy and αSMA immunofluorescence analysis were mounted in 50% glycerol in PBS. For observation of the structure of the anterior or posterior stroma, the epithelial or endothelial side, respectively, of each tissue block was positioned next to the objective lens. The corneal blocks stained for αSMA and nuclei were observed with a laser confocal microscope (LSM 710 NLO; Carl Zeiss, Jena, Germany).