Mda colorimetric fluorometric assay kit

The Lipid Peroxidation (MDA) Assay Kit (Colorimetric/Fluorometric) (Abcam, Cat# ab118970) was used as per the manufacturer’s instructions. Ten eyes per sample were dissected in cold 1×PBS, and then transferred to 50 µL ddH₂O with 1 µL BHT. After homogenization, 50 µL 2 N perchloric acid was added to the slurry. Samples were vortexed and centrifuged to remove precipitated protein. 100 µL of the supernatant was added to 300 µL thiobarbituric acid reagent, and incubated at 95 °C for 1 h. 200 µL of standard or sample were added to individual wells of a GREINER 96 F-BOTTOM plate. For fluorometric measurement, signals were collected with a CLARIOStar reader (BMG LABTECH GmbH) (Ex/Em = 532 ± 8/553 ± 8 nm). Three biological replicates (independent light exposure, paired blue light vs. dark controls) were quantified per sample.

Blood samples collected by cardiac puncture under sterile conditions were centrifuged at 4 °C and 1000 × g for 15 min so that blood plasmas were obtained. Plasma samples frozen rapidly on dry ice were stored at –80 °C until they were used. Lipid peroxidation was determined by measuring malondialdehyde (MDA) levels in plasma samples. In this respect, MDA levels were determined in accordance with the instructions of a commercially available lipid peroxidation (MDA) colorimetric/fluorometric assay kit (BioVision, Milpitas, CA, USA). The absorbance of each sample was measured at 532 nm with an ELISA plate reader (PolarSTAR Omega, BMG LABTECH, Germany) and results were obtained.

Immediately after sacrificing the rats, the liver tissues were dissected and stored at −80°C for detection of antioxidant enzymes. A part of the liver tissues were also excised and fixed for histological staining.
MDA level was measured using lipid peroxidation (MDA) colorimetric/fluorometric assay kit by BioVision, USA. 11β-Hydroxysteroid dehydrogenase enzyme type 1 (11βHSD1) was measured using ELISA kit for 11βHSD1 (Cloud-Clone Corp, USA). Tissue glutathione (GSH) was measured by using glutathione assay kit by Cayman Chemical Company, USA [24 (link)]; glutathione peroxidase (GPx) was measured using glutathione assay kit by Cayman Chemical Company, USA (Forstrom & Wheeler, 1990); catalase (CAT) was measured using catalase assay kit by Cayman Chemical Company, USA [25 (link)]; and superoxide dismutase (SOD) was measured using superoxide dismutase askay Kit by Cayman Chemical Company, USA [26 (link)]. All procedures were done according to the manufacturers' guidelines.

The level of lipid peroxidation activity in the hippocampus and cerebral cortex homogenates was determined by measuring the MDA levels using a lipid peroxidation (MDA) colorimetric/fluorometric assay kit (BioVision, USA, CA) according to the manufacturer’s protocol, as previously described^{58 (link)}. The absorbance was measured at 410 nm using a UV spectrophotometer.

For the determination of oxidative stress parameters and antioxidant component assays in the hippocampal area, the part of stored hippocampal tissue homogenates was used (n = 8 to 10 in each group). All assays were measured by commercial kit.
The lipid peroxidation was determined by the commercial product of Lipid Peroxidation (MDA) Colorimetric/Fluorometric Assay Kit (Catalog#, K739, Bio Vision, Milpitas, CA). The final products of MDA were measured at 530 nm using a spectrophotometer (Soft Max, Ver. 5.4, Molecular Devices, Sunnyvale, CA). Total glutathione (GSH) content was determined using a commercial kit (OxiSelect™, Catalog# STA-312, Cell Bio Labs, INC. San Diego, CA) with the absorbance measured at 405 nm using a spectrophotometer (Soft Max, Ver. 5.4, Molecular Devices). SOD activities were determined using an SOD assay kit (Dojindo Laboratories, Kumamoto, Japan), and dilutions of bovine erythrocyte SOD (St. Louis, MO) ranging in concentration from 0.01 to 50 U/ml were used as standards. Catalase activities were determined using a commercial kit (OxiSelect™, Catalog# STA-314, Cell Bio Labs, INC.). All procedures were carried out, according to the manufacturer's protocol. The absorbance was measured at 450 nm using a spectrophotometer (Soft Max, Ver. 5.4, Molecular Devices).