Mda lysis buffer

The retinas from the intact and illuminated rabbit eyes were isolated in a dim red light at 4 °C immediately after the scarification. Each isolated retina was homogenized for 1 minute on ice in 0.3 mL of 50 mM Tris-HCl, 100 mM NaCl buffer (pH 7.5) using HG-15A 27,000 rpm homogenizer (Witeg Labortechnik, Wertheim, Germany) and the resulting fraction was centrifuged at 24,000× g (16,000 rpm) for 20 min at 4 °C. The supernatants (retinal extracts) were stored at −70 °C until the further studies. The pellets were homogenized in the MDA lysis buffer (Sigma-Aldrich) and the resulting fractions (retinal homogenates) were used for lipid peroxidation measurements.

Mouse brain tissues were homogenized on ice using 300 μl of the MDA lysis buffer according to the manufacturer’s guidance (Sigma-Aldrich, St. Louis, MO) (with 3 μl of butylated hydroxytoluene [100X]), followed by centrifugation (13,000×g, 10 min) to remove insoluble materials. The supernatant (200 μl) from each homogenized sample was placed into a micro-centrifuge tube and incubated with 600 μl of thiobarbituric acid solution at 95 °C for 60 min. Cooled to RT in an ice bath for 10 min, 200 μl of mixtures was pipetted into a 96-well microplate and analyzed by using a fluorescence plate reader (Ex: 532 nm; Em: 553 nm) (Molecular Devices, Sunnyvale, CA).

Full-size rabbit corneas were excised, placed into 400 μl of PBS, and frozen at − 70 °C. After thawing, the tissue was sonicated for 10 min on ice. Corneal extracts for biochemical evaluations were obtained by centrifugation of the samples (15,000 g, 10 min) at + 4 °C. The supernatants were aliquoted and stored at − 70 °C, and the pellets were homogenized in MDA lysis buffer (Sigma-Aldrich) and used for MDA measurements as follows.