Sod 101

Tissue homogenizations in 0.05 M sodium phosphate buffer (pH 6.65) were described in our earlier research (Gavrilović et al., 2012 (link)). The protein concentration was measured by BCA method (Thermo Scientific Pierce, Waltham, MA, USA), described by Stich (1990) (link). Determination of SOD1, SOD2, CAT and GPx was performed using Western blot analysis. Specifically, antibodies used for quantification of proteins were for SOD1 (dilution 1:2,000; SOD-101; Stressgen, Canada), for SOD2 (dilution 1:2,000; SOD-110; Stressgen, Canada), for CAT (dilution 1:2,000; Calbiochem, Germany), GPx (dilution 1:500; sc-30147 Santa CruzBiotechnology, Dallas, TX, USA) and for β-actin (dilution 1:1,000; ab8227, Abcam, USA). A secondary antibody (anti-rabbit, dilution 1:5,000; Amersham ECL™ Western Blotting Analysis System; Amersham, UK) was then visualized by the Western blotting enhanced chemiluminescent detection system (ECL; Amersham Biosciences, Amersham, UK). The result was expressed in arbitrary units normalized in relation to β actin, following our previous protocol (Gavrilović et al., 2013 (link)).

The hippocampus was homogenized in 0.05 M sodium phosphate buffer (pH 6.65). Subsequently, the protein concentration was determined using BCA method (Thermo Scientific Pierce, USA), described by Stich [30 (link)]. CuZn SOD (SOD1), Mn SOD (SOD2), CAT, GPx, GR, BDNF, TH, DAT, and COMT proteins were assayed by Western blot analysis as described previously by Gavrilović et al. [27 (link)]. Antibodies used for the quantification of specific proteins were as follows: SOD1 (SOD-101, Stressgen, USA), SOD2 (SOD-110, Stressgen, USA), CAT (Calbiochem, Germany), GPx (sc-30147 Santa Cruz Biotechnology, USA), GR (sc-32886, Santa Cruz Biotechnology, USA), BDNF (ab6201, Abcam, USA), TH (ab51191, Abcam, USA), DAT (ab18548, Abcam, USA), and β-actin (ab8227, Abcam, USA). After washing, the membranes were incubated in the secondary anti-rabbit (dilution 1 : 5000, Amersham ECL™ Western Blotting Analysis System, UK) antibodies conjugated to horseradish peroxidase. A secondary antibody was then visualized by the Western blotting-enhanced chemiluminescent detection system (ECL, Amersham Biosciences, UK). The membranes were exposed to ECL film (Amersham Biosciences, UK). The result was expressed in arbitrary units normalized in relation to β-actin, which is in accordance with our previous protocol [27 (link)].