Goat anti rabbit and goat anti mouse igg horseradish peroxidase conjugated secondary antibodies

After behavioral testing, 16 rats were anesthetized with chloral hydrate and transcardially perfused with 150 mL of normal saline and 350 mL of 4% paraformaldehyde. Brains were fixed and processed for immunostaining as previously described (Sun et al., 2016). Serial coronal sections (25 μm) were cut through the SNpc (from anteroposterior: −4.8 mm to anteroposterior: −6.2 mm) on a cryostat (Leica, Germany). Free-floating brain sections were pre-incubated in 5% bovine serum albumin containing 0.2% Triton X-100 for 30 minutes at 37°C. These sections were incubated overnight at 4°C with a rabbit polyclonal anti-tyrosine hydroxylase (TH) (1:2000) or mouse monoclonal ionized calcium-binding adaptor molecule-1 (1:200) antibody, both purchased from Millipore (Bedford, MA, USA). Afterwards, sections were incubated with goat anti-rabbit and goat anti-mouse IgG horseradish peroxidase-conjugated secondary antibodies (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA) for 2 hours at room temperature, separately. Stained samples were visualized by diaminobenzidine, and six sections of each animal were counted by two individuals in a blind fashion using a microscope (Olympus, Tokyo, Japan). The survival rate of TH neurons was calculated as the number of TH neurons on the lesioned side relative to the number of TH neurons on the non-lesioned side.

Custom oligonucleotides were synthesized by Sigma Genosys Japan (Ishikari, Japan).
Polyclonal anti-human COMT antibodies (FL-271), monoclonal anti-avian β-actin antibodies
(C4) and goat anti-rabbit and goat anti-mouse IgG horseradish peroxidase conjugated
secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA,
U.S.A.). All other solvents and reagents used were highest grades commercially
available.