Western blotting (WB) analysis
Tissue preparation. An independent group of animals was decapitated and the dorsal striatum was dissected on an ice-cooled dissection cover, with the help of magnifying lens (Leica Zoom 2000), and immediately frozen in dry ice. Tissue samples were stored at −80°C until use. The sample was homogenized on ice in 200uL of tissue in sterile saline using a Polytron®PT 1200 handheld homogenizer (KinematicaInc; NY, USA). The homogenate was used for WB measurement.
Immunoblotting analysis . Protein was isolated from the striatum of both controls and experimental rats. Samples were treated with boiling lysis buffer (1% sodium dodecyl sulfate, 1.0 mM sodium orthovanadate, 10 mM Tris, pH 7.4). Equal amounts (30 μg) of total protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (10%) and transferred to polyvinylidene difluoride membranes. Immunostaining of the blots was performed using two primary antibodies, rabbit polyclonal antibody to NG2 (1:1.000; Millipore) and mouse monoclonal antibody to anti-β-actin (1:10.000; Sigma-Aldrich). Membranes were then incubated with peroxidase-coupled secondary antibodies (1:2.000; Millipore) for 1 hour at room temperature. Blots were developed using the Amersham ECL Prime western blotting detection reagent (GE healthcare, Little Chalfont, UK). Densitometric analysis was performed using the Eagle Eye TMII Still VideoSystem (Stratagene, La Jolla, CA, USA).