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).