Tyrosine hydroxylase immunohistochemistry

After the dissection described above, the posterior part of the brain was immersed overnight in 4% PFA in PBS at + 4 °C, stored in 20% sucrose in PBS at + 4 °C until freezing in isopentane on dry ice, and stored at − 80 °C. Free-floating coronal sections of 30 µm thickness were cut with a Leica CM3050 cryostat (Leica Biosystems, Wetzlar, Germany) and stored at − 20 °C. TH immunostaining was performed essentially as described by Mijatovic et al. [48], with the exception that biotinylated protein A (prepared using protein A [MP Biomedicals, Santa Ana, CA, USA] and N-hydroxysuccinimido-biotin [Sigma-Aldrich]) was used in place of the secondary antibody to reduce background staining. Optical density across the SNC was measured as described previously [21].