4.14. TH Stereological Cell Counting

After the scheduled treatments with rhododendrin (10 mg/kg body weight, daily intraperitoneal administration), animals that received intrastriatal injection with 6-OHDA (intoxication model) or PBS (controls) were anesthetized with pentobarbital (50 mg/kg, intraperitoneal injection) and perfused with PBS. Next, the tissue was fixed with 4% paraformaldehyde (w/v in PBS). Brains were post-fixed overnight with 4% paraformaldehyde and subsequently cryoprotected overnight in 30% sucrose in PBS (w/v). Coronal sections (thickness of 40 µm) were cut through the brain including the substantia nigra. Every fourth section was used for analysis. For analysis of tyrosine hydroxylase (TH) expression, sections were incubated with a 1:1000 dilution of rabbit polyclonal anti-TH (Novus) antibody followed by sequential incubations with biotinylated goat anti-rabbit IgG and streptavidin-conjugated horseradish peroxidase (HRP) using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s instructions. To visualize TH-positive cells, 3,3-diaminobenzidine (DAB, cat# D4293, Sigma) was used as an HRP substrate. Immunostained brain sections were counterstained with Nissl. The total number of TH-positive neurons in the substantia nigra pars compacta was determined using the Optical Fractionator probe in Stereo Investigator software (MicroBrightfield, Williston, VT, USA). All stereological counting was performed in a manner blinded to mouse treatments.