*4.8. Immunohistochemistry*

Hemispheres previously fixed in paraformaldehyde were cryoprotected in 20% sucrose/PBS and embedded in gelatin. Then, sequential coronal brain sections (8 μm thick) near the midstriatum (Bregma 1.00) and SN (Bregma −3.20) were obtained by cryostat sectioning and mounted on SuperFrost-Plus glass slides (Thermo Fisher Scientific). Afterward, sections were incubated in warm PBS at 37 ◦C for 15 min, followed by two washes in PBS, to remove gelatin. Then, sections were blocked in Tris-buffered saline (TBS) containing 10% (*v*/*v*) normal donkey serum (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) and 0.1% (*v*/*v*) Triton X-100 (Sigma-Aldrich) for 1 h. Subsequentially, to stain dopaminergic neurons, sections were incubated with primary rabbit polyclonal antityrosine hydroxylase (TH) antibody (#ab112; Abcam, Cambridge, UK, 1:700), overnight at 4 ◦C. After several washes with PBS, anti-TH primary antibody was detected with diluted (1:200) Alexa Fluor 488 (anti-rabbit) conjugated secondary antibody (Invitrogen— Thermo Fisher Scientific) for 2 h at room temperature. After extensive rinsing, sections were counterstained with Hoechst 33258 (Sigma-Aldrich) and mounted on Mowiol 4-88 (Sigma-Aldrich).
