Polyclonal rabbit anti th

Three days after the last administration of MPTPp, the mice were anesthetized with chloral hydrate (400 mg/kg i.p.), transcardially perfused with 4 % PFA in PB (0.1 M, pH 7.4), and their brains removed and used for immunohistochemistry. Coronal sections (40 μm thick) were cut on a vibratome. Free-floating sections were incubated overnight with TH antibody (polyclonal rabbit anti-TH, 1:1000, Millipore, USA).
The primary antibody was prepared in PBS plus Triton solution containing normal goat serum. After careful washing, the sections were incubated in proper biotinylated secondary antibody (Vector, UK). For visualization, avidin-peroxidase protocol (ABC, Vector, UK) was applied, using 3,3′-diaminobenzidine (Sigma, Italy) as chromogen. After washing, the sections were mounted on gelatin-coated slides, air-dried, dehydrated in ascending concentrations of ethanol, and cleared with xylene [67 (link)].
Adjacent SNc sections were stained with cresyl violet for the Nissl staining to evaluate cell death in this area. For TH and cresyl-violet-stained cells immunohistochemistry in the SNc, three sections were sampled (anterior–posterior: −2.92 to −3.28 mm from bregma) according to the atlas of Paxinos and Franklin 2001. For each mouse, three sections from the striatum (anterior-posterior: 1.10 mm to 0.62 mm from bregma) were analyzed for TH.

After the mouse experiments were completed, the animals were euthanized by injection of 100 mg/kg sodium pentobarbital and then transcardially perfused with 4% paraformaldehyde. Brains were post-fixed in 4% paraformaldehyde and cryoprotected in 30% sucrose and then sectioned using a cryostat. Brain slices were mounted and stained for tyrosine hydroxylase (TH; polyclonal rabbit anti-TH, 1:500) (Millipore, Temecula, CA). Sections were imaged using an Olympus Systems VS120 microscope (Figure 1).