Ber slides and stained with 7.5 mM dihydrorhodamine 123 (DHR) on ice for 45 min, followed by staining with 10 mM Hoechst 33342 for 15 min as described by Wolf et al [42]. The lens in M199 medium was then subjected to confocal image scanning.Image Analysis Using Confocal MicroscopeA laser scanning confocal microscope (Carl Zeiss LSM510 META) was used for whole lens analysis upon vital staining with DNA fluorochrome Hoechst 33342 and ROS marker DHR. The excitation/emission at 405 nm/450 nm was used for Hoechst 3342 and 488 nm/550 nm was used for DHR image. Lens anterior was scanned using 10X objective, and 15900046 65 mm deep into the cortex and 0.8 mm layer Z-scan was performed. The image was analyzed with LSM images analysis software from Zeiss.Western BlotLens fiber fractions (cortex or nucleus) were collected in 50 mM potassium phosphate buffer (pH 7.4), homogenized, and centrifuged. Whole protein extracts were further processed for immunoblot analysis and probed against Gclc using an antiGclc antibody (1:2000; Abnova). All data were normalized to the level of GAPDH and compared with age-matched wild type 
mice.Statistical AnalysisAll values are expressed as means 6 S.D. Statistical significance of difference in mean values was assessed by analysis of variance or Student’s t test. Only p values ,0.05 were considered statistically significant.Lens Protein Enzymatic Digestion for Advanced Glycation Endproduct AnalysisFor AGE analysis by LC/MS, 1 mg of lens protein extract was enzymatically digested in Chelex treated phosphate buffer with sequential additions of peptidase (Sigma P7500), protease K, Methionine enkephalin site pronase and aminopeptidase M (Roche, IN) as described earlier [12]. Corresponding enzyme blanks were incubated without added protein as a background control. Protein concentration was determined using the ninhydrin assay, as described earlier [12].AcknowledgmentsWe thank Christopher Strauch for LC/MC Microcystin-LR web analyses of advanced glycation and oxidation products.Author ContributionsConceived and designed the experiments: XF VMM. Performed the experiments: XF XL SH BW. Analyzed the data: XF VMM. Contributed reagents/materials/analysis tools: MLR. Wrote the paper: XF VMM.
The cornea is our transparent window to the world and its integrity and transparency are essential for proper functioning of the eye. The cornea is a highly 23727046 organised tissue with three distinct cellular layers and two acellular layers. The acellular Descemet’s membrane separates the cellular stroma from the innermost endothelial layer, which is a monolayer of cells in direct contact with the aqueous humour of the anterior chamber. The corneal endothelial layer is responsible for the maintenance of corneal transparency by acting as a “leaky” barrier to allow nutrients to flow from the aqueous humour in the anterior chamber into the collagen stroma and then preventing swelling by actively pumping excess fluid out. This state of equilibrium is lost in disorders such as Fuchs endothelial dystrophy, which is characterised by a progressive oedema of the cornea, due to a loss of endothelial cell density. Fuchs is the most commonly occurring dystrophy in the US affecting approximately 4 of the population over the age of 40 [1]. For treatment of disorders such as Fuchs, severalposterior lamellar techniques have been described as an alternative to the traditional full thickness corneal replacement known as penetrating keratoplasty (PK). These lamellar techniques replace only the defective endothelial lay.Ber slides and stained with 7.5 mM dihydrorhodamine 123 (DHR) on ice for 45 min, followed by staining with 10 mM Hoechst 33342 for 15 min as described by Wolf et al [42]. The lens in M199 medium was then subjected to confocal image scanning.Image Analysis Using Confocal MicroscopeA laser scanning confocal microscope (Carl Zeiss LSM510 META) was used for whole lens analysis upon vital staining with DNA fluorochrome Hoechst 33342 and ROS marker DHR. The excitation/emission at 405 nm/450 nm was used for Hoechst 3342 and 488 nm/550 nm was used for DHR image. Lens anterior was scanned using 10X objective, and 15900046 65 mm deep into the cortex and 0.8 mm layer Z-scan was performed. The image was analyzed with LSM images analysis software from Zeiss.Western BlotLens fiber fractions (cortex or nucleus) were collected in 50 mM potassium phosphate buffer (pH 7.4), homogenized, and centrifuged. Whole protein extracts were further processed for immunoblot analysis and probed against Gclc using an antiGclc antibody (1:2000; Abnova). All data were normalized to the level of GAPDH and compared with age-matched wild type mice.Statistical AnalysisAll values are expressed as means 6 S.D. Statistical significance of difference in mean values was assessed by analysis of variance or Student’s t test. Only p values ,0.05 were considered statistically significant.Lens Protein Enzymatic Digestion for Advanced Glycation Endproduct AnalysisFor AGE analysis by LC/MS, 1 mg of lens protein extract was enzymatically digested in Chelex treated phosphate buffer with sequential additions of peptidase (Sigma P7500), protease K, pronase and aminopeptidase M (Roche, IN) as described earlier [12]. Corresponding enzyme blanks were incubated without added protein as a background control. Protein concentration was determined using the ninhydrin assay, as described earlier [12].AcknowledgmentsWe thank Christopher Strauch for LC/MC analyses of advanced glycation and oxidation products.Author ContributionsConceived and designed the experiments: XF VMM. Performed the experiments: XF XL SH BW. Analyzed the data: XF VMM. Contributed reagents/materials/analysis tools: MLR. Wrote the paper: XF VMM.
The cornea is our transparent window to the world and its integrity and transparency are essential for proper functioning of the eye. The cornea is a highly 23727046 organised tissue with three distinct cellular layers and two acellular layers. The acellular Descemet’s membrane separates the cellular stroma from the innermost endothelial layer, which is a monolayer of cells in direct contact with the aqueous humour of the anterior chamber. The corneal endothelial layer is responsible for the maintenance of corneal transparency by acting as a “leaky” barrier to allow nutrients to flow from the aqueous humour in the anterior chamber into the collagen stroma and then preventing swelling by actively pumping excess fluid out. This state of equilibrium is lost in disorders such as Fuchs endothelial dystrophy, which is characterised by a progressive oedema of the cornea, due to a loss of endothelial cell density. Fuchs is the most commonly occurring dystrophy in the US affecting approximately 4 of the population 
over the age of 40 [1]. For treatment of disorders such as Fuchs, severalposterior lamellar techniques have been described as an alternative to the traditional full thickness corneal replacement known as penetrating keratoplasty (PK). These lamellar techniques replace only the defective endothelial lay.
