Tyrosine Hydroxylase Antibody

The tyrosine hydroxylase antibody was successfully used for immunostaining of dopamine neurons in the adult Drosophila brain. The antibody performed very well for this application. Drosophila brains were collected and stained as follows: 1. Dissected in PBT buffer 2. Fixed in 4% PFA for 20 min at RT 3. Washed in PBT twice 4. Blocked in 5% normal goat serum in PBT buffer for 30 min at RT 5. Incubated in anti-TH primary (1:1000) for 48 h at 4 deg C 6. Washed 5 times in PBT 7. Incubated in anti-mouse alexa fluor-488 secondary antibody for 48h at 4 deg C 8. Washed 5 times in PBT before mounting and imaging Dopamine neuron cell bodies and projections within the canonical anterior and posterior clusters could be readily visualized by confocal microscopy z-stack imaging. There was very little background staining in the Drosophila brain.

I have used the mouse antibody for tyrosine hydroxylase (Product ID : 22941) for staining dopaminergic cells in Drosophila melanogaster adult brains, larval brains, as well as primary neuronal cultures, and have always had consistent results, as well as a good signal-to-noise ratio of 4 to 1. In published results, I compared the staining of this antibody with a GFP signal driven by tyrosine hydroxylase and the overlap between the two signals was good (J Neurochem. 2013 Aug;126(4):529-40. doi: 10.1111/jnc.12228. Epub 2013 Mar 24). Also, in unpublished results, it appeared to perform just as well as a Drosophila antibody for tyrosine hydroxylase. I have used this antibody for years and have always obtained consistent results.

We have used the ImmunoStar Inc. tyrosine hydroxylase antibody (22941) with great success, for identifying TH-IR cells in the striatum and midbrain of European starling (Sturnus vulgaris). We typically use it at a 1:3000 dilution, which produces distinct staining with very low background. Tissue preparation: Birds are transcardially perfused with 1X phosphate-buffered saline (PBS), followed by 4% paraformaldehyde. The brain is extracted and post-fixed in 4% paraformaldehyde overnight, then transferred to 30% sucrose for 2-3 days (until the brain sinks). Brains are sectioned at 40 µm on a sliding microtome equipped with a freezing stage, and collected into PBS. Immunohistochemistry procedure: Brain sections are transferred to 24-well plates. All solutions are made up in 1X PBS and are typically applied at a volume of 0.5 ml. The plates are gently agitated on a shaker during incubations. DAY 1 • Rinse in PBS 3 times, for 10 min each • Incubate in 3% hydrogen peroxide for 10 min • Rinse in PBS 3 times, for 10 min each • Incubate in blocking serum (from Vector ABC Elite kit) + 0.2% Triton X-100 for 30 min • Incubate in primary antibody + 0.5% Triton X-100 overnight at 4°C DAY 2 • Rinse in PBS 3 times, for 10 min each • Incubate in secondary antibody (from Vector ABC Elite kit) + 0.6% Triton X-100 for 30 min • Rinse in PBS 3 times, for 10 min each • Incubate in ABC reagent (from Vector ABC Elite kit) + 0.6% Triton X-100 for 30 min • Rinse in PBS 3 times, for 10 min each • Incubate in 0.05% DAB (3’3-diaminobenzidine) + 0.015% hydrogen peroxide for 2-3 min • Rinse in PBS 3 times, for 10 min each Float-mount sections onto gelatin-subbed slides and let dry overnight, then dehydrate with ethanol and xylene rinses and coverslip with Permount.