Ecl film

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)].

Cells were lysed in Laemmli sample buffer [2% SDS, 5% glycerol, 1.5% dithiothreitol (DTT), 0.01% Bromophenol Blue and 60 mM Tris-HCl pH 6.8]. Collected cells were sonicated (Diagenode) with three bursts of 15 s and heated for 10 min at 95°C. 10–15 μl of lysate was loaded on an SDS–polyacrylamide gel with a width of 1 mm, along with 7 μl of molecular weight markers (Bio-Rad). A voltage of 60 V for the stacking gel and 150 V for the resolving gel were applied. Gels were run in Tris-glycine electrophoresis buffer (25 mM Tris, 250 mM glycine and 0.1% SDS). For western blotting analysis, proteins were transferred to a 0.2 μm nitrocellulose membrane using the Trans-Blot Turbo Transfer System apparatus (Bio-Rad). The transfer was performed at 25 V for 7 or 10 min (according to the molecular weight of the proteins under investigation). Membranes were incubated with 5% skim milk in TBS-T buffer (TBS containing 0.1% Tween-20) for 1 h, followed by overnight incubation at 4°C with primary antibody and three washes with TBS-T before 1 h incubation at room temperature with the specific HRP-conjugated secondary antibody. Chemiluminescence detection was done by incubation with Luminata Classico or Crescendo (Millipore). Proteins were visualized by autoradiography on ECL films (Amersham) using various exposure times and manual development, or by using a Chemidoc imaging system (Bio-Rad).