The average diameter of the beads was estimated at 35 μm. For the control batch the procedure was similar except the addition of rotenone. Immunohistochemistry Cryo-embedded brains were cut on a cryostat (30 μm thickness) and collected on Superfrost slides. The slices were dried in a 42°C oven during 18 h then stored at −20°C. Immunohistochemistry experiment required the use of an antigen Inhibitors,research,lifescience,medical retrieval method. The antigen retrieval

was performed in a commercial microwave oven (1600 watts). The slides were placed in a preboiled solution of 1 mM EDTA (sellectchem ethylenediaminetetraacetic acid), 10 mM Tris-Cl, pH 8 and microwaved for 15 min at 20% of the maximum power of the oven (80–95°C). The solution was cooled to room temperature and Inhibitors,research,lifescience,medical the slides transferred to phosphate buffered saline (PBS) for the staining procedures. Brain slices were washed in PBS two times during 5 min and incubated in blocking reagent (PBS pH 7.8, 10% FBS (fetal bovine serum), 0.1% triton X-100) for 2 h. The appro-priate primary antibody was applied Inhibitors,research,lifescience,medical over night at 4°C in the blocking solution (NeuN 3 μg/mL, VMAT2 2.5 μg/mL, DAT 3 μg/mL, TH 2.5 μg/mL, Ubiquitin 3 μg/mL, α-synuclein 3 μg/mL, GFAP 1.25 μg/mL, microglia CD11b 3 μg/mL). After three washes in PBS secondary antibodies were incubated at room temperature for 4 h. For fluorescent staining, the slides were mount with Vectashield (Vector Lab., Burlingame, CA). For diaminobenzidine Inhibitors,research,lifescience,medical (DAB)

staining, we used biotinylated secondary antibodies Enzastaurin chemical structure revealed by the ABC kit (Vector Lab.). The slides were then

counterstained with cresyl violet, dehydrated, and mounted with Permount (Fisher). Note, for DAB staining the slides were preincubated in methanol 3% hydrogen peroxide (H2O2) for 20 min before the blocking step. The 7,8-dihydro-8-oxo-deoxyguanine (8-oxo-dG) staining was performed as previously described by Marella et al. (2007). Briefly, brain slices were treated with RNase A, then, after an incubation in 4 N HCl the acid was neutralized and the slices were blocked for immunostaining. The determination of iron accumulation in SN was done Inhibitors,research,lifescience,medical by a method largely inspired by Nguyen-Legros et al. (1980), as a new histochemical demonstration Batimastat of exogenous iron. The brain sections were immersed in a Perl’s staining solution of 5% HCl, 10% potassium ferrocyanide in water at room temperature during 1 h. After three washes with ultrapure distilled water the sensitivity of the staining was increased by secondary reactions with DAB and H2O2 for 20 min. The slices were counterstained with cresyl violet, dehydrated, and mounted with Permount. For SPECT/CT imaging animals were anesthetized during i.v. administration of 125I-betaCIT (0.4 mCi, 0.3 mL) and were returned to their cages after injection for the uptake period. In vivo images were acquired at 3 h postinjection using the NanoSPECT/CT® (Bioscan, Washington, DC).