Mouse monoclonal anti th antibody

Following transcardial perfusion with fixative, the brains of each rat were removed and post-fixed overnight in 4% paraformaldehyde (4°C) and then placed in 30% sucrose in 0.1 M phosphate saline buffer (PBS) at 4°C until the brains sank. A one in ten series of 20 μm sections were cut through from −5.8 to −8.8 mm from bregma using a freezing microtome (Leica, Germany). All sections were treated identically and were reacted at each stage, for exactly the same time. Sections were incubated in 3% H₂O₂ to block endogenous peroxidase activity, blocked in 10% normal horse serum (NHS) and 0.3% triton-X in PBS and then incubated in a mouse monoclonal anti-TH antibody (1:10,000, ImmunoStar) at 4° overnight. Sections were washed in PBS then incubation in biotinylated donkey anti-mouse IgG (1:500; Jackson Immunochemicals, West Grove, PA, USA) for 2 h. Following a wash in PBS, the sections were then incubated in ExtrAvidin peroxidase (1:1,000; Sigma-Aldrich, St. Louis, MO, USA) for 2 h. The bound antibody complex was visualised using DAB (0.05%) as the chromagen. Sections were mounted onto gelatinized slides; allowed to dry overnight; dehydrated through graded alcohols and then coverslipped with DPX mounting medium.

Protein was isolated from the organic phase in the TRI reagent protocol. Total protein concentrations for each midbrain block were estimated using the Direct Detect™ Spectrometer (Millipore, Bedford, MA, USA). 15 μg of total protein was then separated on 12% Criterion TGX stain-Free gels (Bio-Rad) and transferred to PVDF membranes using the trans-Blot^® Turbo Transfer packs (Bio-Rad). Membranes were blocked in skim milk powder (5%) and incubated overnight at 4°C with a mouse monoclonal anti-TH antibody (1:10,000, ImmunoStar), followed by incubation with HRP-conjugated goat anti-mouse IgG (1:5,000, Santa Cruz). TH was visualised using the Immobilon Western chemiluminescence HRP substrate (Millipore, Bedford, MA, USA) on a ChemiDoc MP imaging system (Bio-Rad). Bands were quantified using Image Lab™ software.
Membranes were then incubated in rabbit polyclonal anti-beta actin antibody (1:1,000, Santa Cruz) followed by HRP-conjugated goat anti-rabbit IgG (1:5,000, Santa Cruz). The labeled beta actin was visualised and quantified as described above.

Immunohistochemistry was performed on Drosophila adult brains expressing cytosolic GFP (UASGFP) with the specified GAL4 strains, after fixing the dissected tissue in 4% paraformaldehyde. The following primary antibodies were used: mouse monoclonal anti-TH antibody (1:50, #22941, ImmunoStar, Hudson, WI, USA), rabbit anti-GFP antibody (1:10,000; #A6455, Molecular Probes, Eugene, OR, USA), mouse anti-Fz2 (1:20; #12A7, DSHB, University of Iowa). 12A7 was deposited to the DSHB by Nusse, R (DSHB Hybridoma Product 12A7). Fluorescent secondary antibodies were used at a dilution of 1:400 as follows: anti-rabbit Alexa Fluor 488 (#A1108) and anti-mouse Alexa Fluor 568 (#A1104, Molecular Probes, Eugene, OR, USA). After antibody staining, confocal analysis was performed on an Olympus Confocal FV1000 microscope and visualized using the FV10-ASW 1.3 viewer (Olympus Corporation, Tokyo, Japan).