For quantification of CML content, retinal samples were subjected to ultrasonic disruption in RIPA buffer. After centrifugation to remove tissue debris, the protein content of the resultant supernatant was measured with a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL). Fifty microliters of sample (concentration: μg/mL) or advanced glycation end product (AGE)-BSA standard (0–200 μM) diluted in 0.05% Tween 20 and 0.2% BSA in 75 mM PBS were aliquoted in duplicate into a plate (Nunc C96 Maxisorp; eBioscience, San Diego, CA) that had been precoated with solid-phase antigen (1 μg/mL AGE-BSA in 0.05 M carbonate buffer [pH 9.6]) overnight at 4°C and blocked with 3% skim milk powder for 1.5 hours. Fifty microliters of 1:2000 rabbit polyclonal anti-CML antibody (gift from Suzanne Thorpe, Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC) was added to sample and standard wells, and plates were incubated for 2 hours at room temperature. Wells containing 100 μL of diluting buffer were used as a nonsample, nonantibody blank. Plates were washed in buffer (0.2 mM KH2PO4, 1.4 mM K2HPO4, 3 mM NaCl, 0.45 μM sorbic acid potassium, and 0.01% Tween-20) using an ELISA plate washer (3 × 300 μL) and 100 μL of peroxidase-conjugated anti-rabbit IgG (diluted 1:5000 in 0.1% BSA in PBS; Sigma-Aldrich) was added to each well and incubated for 1 hour at room temperature. Plates were again washed before the color reaction was started by the addition of 150 μL of tetramethyl-benzine substrate (TMB; Sigma-Aldrich) to each well. The reaction was stopped after 30 minutes by the addition of 50 μL of 2 M sulfuric acid, and the absorbance of the blue reaction product was measured at 450 nm on a microplate reader (Safire; Tecan Instruments, Switzerland). Results (minus blank absorbance) were expressed as a percentage of the zero standard (nonsample, antibody control) and CML concentration in samples determined by comparison to the standard curve.