Mouse anti tyrosine hydroxylase

We perfused mice transcardially with PBS followed by 4% paraformaldehyde, and soaked the brains in 30% sucrose. We sectioned OBs coronally (30 µm) on a sliding microtome and performed immunohistochemsitry on floating sections in 5% heat-inactivated goat serum and 0.5% triton in PBS. We used the following primary antibodies: rabbit anti-calretinin (CR) (1:2000) and mouse anti-calbindin (CB) (1:1000) (Swant, Bellinzona, Switzerland), mouse anti-Tyrosine Hydroxylase (TH) (1:500) (Immunostar, Hudson, WI, USA), mouse anti-Tbr2 (1:100) (Abcam, Bristol, UK) and mouse anti-NeuN (Millipore, Billerica, MA, USA). GCaMP was amplified using chicken anti-GFP (1:1000, Millipore). The following secondary antibodies were used: biotinylated goat anti-rabbit (1:500), DyLight549 conjugated goat anti-mouse (1:500) and DyLight488 conjugated goat anti-chicken, (Jackson ImmunoResearch, West Grove, PA, USA). Amplification was carried out using Cy5 conjugated streptavidin (Jackson ImmunoResearch). Slices were imaged with a SP50 confocal microscope, via a 60X (1.4 NA) oil objective (Leica, Wetzlar, Germany). Counting of neuronal somata was carried out manually using ImageJ.

Most tissue preparation and immunostainings were performed as described previously (21 (link)) For DAB stainings in this study, the following primary antibodies were used: mouse anti-tyrosine hydroxylase (TH) (1:1,000, Immunostar, Hudson, WI, USA), rabbit anti-vesicular monoamine transporter 2 (VMAT2) (1:4,000, Immunostar Hudson, WI USA), mouse anti-human WT α-syn (1:2,000, Santa Cruz, CA, USA), and chicken anti-GFP (1:20,000 Abcam, Cambridge, UK). The SNpc sections were given an initial antigen-retrieval incubation in Tris/EDTA (pH 9.0) at 80°C for 45 min when stained for TH.
Double immunofluorescence stainings were performed as described previously (21 (link)). The primary antibodies used were rabbit anti-GSTA4 (1:100 Antibodies-online GmbH, Aachen, Germany), mouse anti-Gfap (1:1,000, Santa Cruz, CA USA), chicken anti-IBA1 (1:500 Synaptic Systems, Göttingen, Germany), and mouse anti-NeuN (1:1,000 Millipore, Billerica, MA USA) and were incubated together at 4°C. To compare immunofluorescent stainings of midbrain and striatum for Gsta4 and Gfap at 3 and 8 weeks, stainings were performed in parallel and images were taken with the same settings. All images were captured at high-resolution with the confocal Leica SP8 microscope (Leica Microsystems, Wetzlar, Germany).

For analysis of muscle, thoraces of 1- to 2-day-old-adult flies were dissected and fixed in 4% paraformaldehyde in phosphate buffered saline (PBS). After thoraces were washed three times in PBS, muscle fibers were isolated and stained with rhodamine phalloidin (Invitrogen, 1:1000) in PBS+1% Triton X-100. For antibody staining, muscle fibers were permeabilized in PBS+0.1% Triton X-100, blocked in 5% normal goat serum in PBS, and incubated in primary and secondary antibodies diluted in 5% normal goat serum in PBS. For analysis of dopaminergic neurons, brains of 3-day-old male flies were dissected and fixed in 4% paraformaldehyde in PBS. Blocking, primary and secondary antibody staining were performed as described previously (Yun et al., 2008 (link)). To analyze mitochondria in salivary glands, salivary glands of third instar larvae were dissected, fixed in 4% paraformaldehyde in PBS, and stained with rhodamine phalloidin. The following primary antibodies were used: mouse anti-ATP Synthase (Mitosciences, Eugene, OR), chicken anti-HA (Millipore, Billerica, CA), mouse anti-Tyrosine Hydroxylase (Immunostar Hudson, WI). All images were taken on a Zeiss LSM5 confocal microscope.