2.4. Analysis of lipid peroxidation (MDA)

Lipid peroxidation was measured by TBARS Assay Kit (OxiSelect TBARS Assay Kit, CellBiolabs). The principle of this method is based on the formation of a 1:2 conjugate of Malondialdehyde (MDA) with thiobarbituric acid (TBA). First, the tissue samples were diluted with 5% butyl hydroxytoluene, and 100 μL of sodium dodecyl sulfate (SDS) lysis solution was added to 100 μL of the sample and incubated at room temperature for 5 min. After that, 250 μL TBA solution was added to samples, and the mixtures were incubated at 95°C for 45 min. Thereafter, the mixtures were cooled on ice for 5 min and centrifuged at 10.000 x g for 15 min. Then, 200 μL of the supernatant was taken and transferred to a new tube, and 300 μL butanol was added. The mixture was vortexed at 3000 rpm for 3 min and centrifuged at 10.000 x g for 5 min. The absorbances of the supernatants were read at a wavelength of 532 nm in a microplate reader (SpectraMax M2, Molecular Devices, United States). A standard graph was employed to calculate the results. The results were standardized by dividing the wet tissue weight.