p; 50 mg/kg; Cristalia, Brazil) Heparin (1000 IU; Cristalia, Bra

p; 50 mg/kg; Cristalia, Brazil). Heparin (1000 IU; Cristalia, Brazil) was injected into the left cardiac ventricle, then the animals were transcardially perfused through the left ventricle using a peristaltic pump (Control Company, Brazil, 20 mL/min) with 400 mL of 0.9% saline solution, followed by 400 mL of a fixative solution 4% GDC-0449 purchase paraformaldehyde (Synth, Brazil) in 0.1 M phosphate buffer, pH 7.4 (PB). The brains were removed from the skulls, post-fixed in the same solution at room temperature for 4 h and cryoprotected by immersion in a 15% and 30% sucrose (Synth, Brazil) solution in PB at 4 °C until they sank. After these procedures, the brains were quickly frozen in isopentane (Merck, Germany) cooled in liquid nitrogen and kept in a

freezer (−70 °C) for further analyses. Coronal sections (50 μm) from VTA and SNpc were obtained from each brain using a cryostat (CM1850, Leica, Germany) at −20 °C and collected in a PB saline (PBS), pH 7.4. These areas were identified using Paxinos and Watson’s Atlas (1998). The free-floating sections were pre-treated with 3% hydrogen peroxide for

30 min, carefully washed and treated with 2% bovine serum albumin (Inlab, Brazil) in PBS containing 0.4% Triton X-100 (PBS-Tx) for 30 min and incubated with monoclonal TH antibody (Sigma Chemical Co., USA) raised in mice, diluted 1:2000 in PBS-Tx for 48 h at 4 °C. Sections were again washed in PBS-Tx and incubated in an anti-mouse antibody conjugated with peroxidase (Sigma Chemical Co., Selleck AZD6244 USA) diluted 1:200 in PBS-Tx for 2 h at room temperature. The reaction was revealed in a medium containing 0.06% 3,3′-diaminobenzidine (DAB, Sigma Chemical Co., USA) dissolved in PBS for 10 min and

then 1 μL of 3% H2O2/mL was added to the DAB medium for an additional 10 min. Finally, the sections were rinsed in PBS, dehydrated in ethanol, cleared with xylene and covered with Entellan (Merck, Germany) and coverslips. Control sections were prepared omitting the primary antibody by replacing it with PBS. Semi-quantitative densitometric analysis was used to measure the intensity of the TH immunoreaction using a Nikon Optiphot-2 microscope (40×, Japan) coupled to a Micrometrics camera (Accu Scope, only USA) and Image Pro Plus Software 6.0 (Media Cybernetics, USA). The digitized images obtained from the selected areas were converted to an 8-bit gray scale (0–255 gray levels). All lighting conditions and magnifications were held constant. Picture elements (pixels) employed to measure optical density were obtained from squares measuring 9680 μm2 (area of interest, AOI) overlaid on the gray scale image. Both left and right sides of each brain were used. For each rat, 10 measures were taken from the VTA and 10 measures each from the medial, lateral and intermediary regions of the SNpc. The results shown for the SNpc were the total mean value from the three studied regions. Background staining subtraction and correction were done in accordance with our previous published protocol (Xavier et al., 2005).

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