Monoclonal mouse anti th antibody

To visually confirm the success of 6-OHDA infusion, immunostaining for tyrosine hydroxylase (TH) was performed at the striatum and substantia nigra [40 (link)]. All experimental animals were perfused with 4% paraformaldehyde and then, brains were postfixed in the perfusion solution and cryoprotected in 25–30% sucrose. Coronal sections 40 μM thick were prepared with a freezing microtome and serial sections were processed immunohistochemically. Staining for TH occurred by incubation of a monoclonal mouse anti-TH antibody (Sigma-Aldrich; #T1299, 1:250), followed by incubation of a biotinylated goat anti-mouse secondary antibody (Vector Laboratories, Burlingame, California, USA, 1:200). Sections were amplified with the avidin-biotin complex method (Vectastain Elite ABC‐HRP Kit; Vector Laboratories) for 1 and detected by the 3,3’-diaminobenzidine method (Sigma-Aldrich). This qualitative verification of 6-OHDA infusion by TH staining has been previously described in detail by our group [40 (link)].

The animals (n = 30) were deeply anesthetized through immersion in carbonate-buffered tap water containing MS-222 (100 mg/l; Sigma, St Louis, MO, USA). Following decapitation, a portion of the spinal cord, rostral to the dorsal fin, was fixed by 4% (wt/vol) paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 12–24 hr at 4°C, and subsequently cryoprotected in 20% sucrose in phosphate buffer (PB) for 3–12 hr. For GABA and dopamine immunodetection, 1% glutaraldehyde (vol/vol) was added to the fixative solution. Transverse sections (20 µm thick) were cut on a cryostat (Microm HM 560, Microm International GmbH, Walldorf, Germany), collected on gelatine-coated slides, and kept at −20°C until processing. Sections were incubated/co-incubated with different primary antibodies listed here: a mouse monoclonal anti-acetylated tubulin antibody (dilution 1:500, Sigma-Aldrich), a rat monoclonal anti-somatostatin antibody (1:100, Millipore), a rabbit polyclonal anti-somatostatin-14 IgG antibody (1:1000, Peninsula laboratories), a mouse monoclonal anti-TH antibody (1:200, Millipore), a rabbit polyclonal anti-TH antibody (1:500, Millipore), a mouse monoclonal anti-dopamine antibody (1:400, Millipore), and a mouse monoclonal anti-GABA antibody (1:2000, Swant).