Rabbit anti th polyclonal antibody

NAPS and SCH442-416 were purchased from Tocris Bioscience (Ellisville, MI, USA). Lipofectamine 2000 was from Invitrogen (Carlsbad, CA, USA). SNAP-Lumi4-Tb (GK), NAPS-Lumi4-Tb and SCH-red were from Cisbio Bioassays (Bagnols-sur-Cèze, France). The primary antibodies used were: rabbit anti-TH polyclonal antibody (Millipore, Temecula, CA, USA), goat anti-A_2AR and rabbit anti-D₂R polyclonal antibodies (Frontier Institute Co. Ltd, Shinko-nishi, Ishikari, Hokkaido, Japan).

In a different group of mice 30 μm horizontal sections were cut with a cryostat (Leica, Germany), collected in parallel series, and immersed for 30 minutes in 3% H₂O₂ to inactivate endogenous peroxidase, and incubated for 60 minutes at room temperature in 4% normal goat serum (NGS; Jackson ImmunoResearch, West Grove, PA) in PBS, containing 0.05% Triton X-100 (TX-100; Sigma), and overnight in PBS containing 2% NGS and rabbit anti-TH polyclonal antibody (1: 3000; Millipore, USA). After several rinses, the sections were incubated for 2 hours in biotinylated rabbit anti-goat antiserum (1:300; Sigma), and 1:200 NGS in PBS. Immunoreactions were visible after incubation for 1 hour at RT in ExtrAvidin–peroxidase (1:1500; Sigma) in PBS, and after 5 minutes in 0.005% 3′-3′-diamiobenzidine tetrahydrochloride (DAB; Sigma) and 0.001% H₂O₂ in cacodylate buffer 0.05 N pH 7.6. After several rinses, sections were mounted on gelatinized slides, dehydrated, coverslipped with DePeX and photographed with a Leica microscope (Leica, N.A. 1.36).

Triglyceride of docosahexaenoic acid (TG-DHA) obtained by enzymatic synthesis was kindly supplied by Brudy Technology (Barcelona, Spain). This oil contains more than 70% TG-DHA and more than 90% of ω-3 of total fatty acids (Mancera et al., 2017 (link)). The stock TG-DHA solution in 100% ethanol was diluted with the indicated aqueous buffer before use. All other compounds were obtained from external sources: 6-hydroxydopamine (6-OHDA) (Sigma-Aldrich, St. Louis, MO, United States) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium (MTT) (Sigma-Aldrich). The primary antibodies used were rabbit anti-TH polyclonal antibody (Millipore, Temecula CA, United States) and rabbit anti-α-actinin-1 polyclonal antibody (Santa Cruz biotechnology, Dallas TX, United States). The secondary antibodies used were horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Pierce Biotechnology, Rockford, IL, United Staes) and Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, United States).