Oxidative Stress and Antioxidant Evaluation in Brain

For the determination of oxidative stress parameters and antioxidant components in the brain, frozen tissues were homogenized in RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with Complete and Phospho STOP (Roche, Basel, Switzerland) protease and phosphatase inhibitors as described [20 (link)]. Lipid peroxidation was determined using a commercial TBARS assay kit (CA995, Canvax, Cordoba, Spain). The final malondialdehyde products were detected by fluorescence spectroscopy with excitation/emission at 530 nm/590 nm in a microplate reader (POLARstar Omega, BMG Labtech, Ortenberg, Baden-Wuerttemberg, Germany). Levels of superoxide dismutase (SOD) activity were determined using an SOD assay kit (CA061, Canvax, Córdoba, Spain), according to the manufacturer’s protocol. Absorbance at 450 nm was measured using a POLARstar Omega plate reader. Catalase activities were determined using a commercial Catalase Activity assay kit (CA063, Canvax, Córdoba, Spain) following manufacturer’s instructions. Enzyme activity was detected by fluorescence spectroscopy with excitation/emission at 530 nm/590 nm in a microplate reader (POLARstar Omega).