The blot was washed twice again for 5 min with T-TBS and twice for 5 min with TBS. The blot was then developed using a chemiluminescence ECL kit. Immunoblots were quantified by scanning the films with a Hewlett-Packard Scanjet 6100C scanner and determining optical densities with an OptiQuant version 02.00 software (Packard Instrument Company). Optical density values were obtained for the studied proteins. RNA

was isolated from striatum OSI-744 research buy using the TRIzol Reagent (Invitrogen). Approximately 2 μg of total RNA were added to each cDNA synthesis reaction using the SuperScript-II RT pre-amplication system. Reactions were performed at 42 °C for 1 h using the primer T23 V (5′ TTT TTT TTT TTT TTT TTT TTTTTV). Quantitative

PCR amplification was carried out using specific primer pairs designed with Oligo Calculator version 3.02 (http://www.basic.nwu.edu/biotools/oligocalc.html) and synthesized by IDT (MG, Brazil). The sequences of the primers used are listed in Table 1. Quantitative PCRs were carried out in an Applied-Biosystem StepOne Plus real-time cycler and done in quadruplicate. Reaction settings were composed of an initial denaturation step of 5 min at 95 °C, followed by 40 cycles of 10 s at 95 °C, 10 s at 60 °C, 10 s at 72 °C; samples were kept for 1 min at 60 °C for annealing and then heated from 55 to 99 °C with a ramp of 0.3 °C/s to acquire data to produce the denaturing curve of the amplified products. Quantitative PCRs were made in a 20 μl final volume composed of 10 μl of each reverse transcription sample diluted 50–100 LDK378 supplier times, 2 μl of 10 times PCR buffer, 1.2 μl of 50 mM MgCl2, 0.4 μl of 5 mM dNTPs, 0.8 μl of 5 μM primer pairs, 3.55 μl of water, 2.0 μl of SYBRgreen (1:10,000 Molecular Probe), and 0.05 μl of Platinum Taq mafosfamide DNA polymerase

(5 U/μl). All results were analyzed by the 2 − DDCT method (Livak and Schmittgen, 2001). TBP (TATA box binding protein) was used as the internal control gene for all relative expression calculations. Twelve pups (six per group) were anesthetized using ketamine/xylazine (75 and 10 mg/kg, respectively, i.p.) and were perfused through the left cardiac ventricle with 40 ml of 0,9% saline solution, followed by 40 ml of 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS), pH 7.4, and the descendent aorta was clamped. After the perfusion the brains were removed, post-fixed in the same fixative solution for 4 h at room temperature and cryoprotected by immersing in 15% and after in 30% sucrose solution in PBS at 4 °C. The brains were then frozen by immersion in isopentane cooled with CO2 and stored in a freezer (− 80 °C) for later analyses. Serial coronal sections (40 μm) of striatum were obtained using a cryostat at − 20 °C (Leica).