Tyrosine Hydroxylase / TH (HMV312)

The enzyme tyrosine hydroxylase (TH) or tyrosine 3-monooxygenase is coded by the TH gene at 11p15.5. TH catalyzes the rate limiting step in the synthesis of catecholamines which is the conversion of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA). L-DOPA is a precursor for dopamine, which, in turn, is a precursor for the important neurotransmitters norepinephrine (noradrenaline) and epinephrine (adrenaline). These catecholamines play important roles in a variety of physiological and behavioral functions in the nervous and endocrine systems. Tyrosine hydroxylase deficiency leads to impaired synthesis of catecholamines and causes a progressive encephalopathy with poor prognosis. TH is also involved in other neurological diseases. For example, cerebral TH activity is significantly reduced in patients with Alzheimer’s disease as compared to healthy individuals. Tyrosine hydroxylase is also an autoantigen in Autoimmune Polyendocrine Syndrome (APS) type I.

In normal tissues, TH is present in specific regions of the central nervous system (CNS), peripheral sympathetic neurons and nerve fibers, as well as in the adrenal medulla. TH is mainly present in the cytosol, although it also is found in the plasma membrane probably due to catecholamine packing in vesicles and export through the synaptic membrane. Images describing the Tyrosine Hydroxylase (TH) staining pattern in normal tissues obtained by the antibody HMV312 are shown in our “Normal Tissue Gallery”.

These findings are largely consistent with the RNA data described in the Human Protein Atlas (Tissue expression TH). 

Positive control = Adrenal gland: A strong cytoplasmic and membranous TH staining should be seen in medullary cells as well as in a subset of nerve fibers in the cortex.

Negative control = Adrenal gland: TH staining must be absent in adrenocortical cells.

Normal tissue gallery

Cancer tissue gallery

No data available at the moment

IHC users have different preferences on how the stains should look like. Some prefer high staining intensity of the target stain and even accept some background. Others favor absolute specificity and lighter target stains. Factors that invariably lead to more intense staining include higher concentration of the antibody and visualization tools, longer incubation time, higher temperature during incubation, higher temperature and longer duration of the heat induced epitope retrieval (slide pretreatment). The impact of the pH during slide pretreatment has variable effects and depends on the antibody and the target protein.

All images and data shown here and in our image galleries are obtained by the manual protocol described below. Other protocols resulting in equivalent staining are described as well.

Manual protocol

Freshly cut sections should be used (less than 10 days between cutting and staining). Heat-induced antigen retrieval for 5 minutes in an autoclave at 121°C in pH 7,8 Target Retrieval Solution buffer. Apply HMV312 at a dilution of 1:150 at 37°C for 60 minutes. Visualization of bound antibody by the EnVision Kit (Dako, Agilent) according to the manufacturer’s directions.

• Quantification of TH in brain and nerve tissues.
• The clinical significance of the level of TH expression in paragangliomas needs to be further evaluated.
• The specificity of TH immunostaining for pheochromocytoma and paraganglioma needs to be determined.

There are two ways how the specificity of antibodies can be documented for immunohistochemistry on formalin fixed tissues. These are: 1. Comparison with a second independent method for target expression measurement across a large number of different tissue types (orthogonal strategy), and 2. Comparison with one or several independent antibodies for the same target and showing that all positive staining results are also seen with other antibodies for the same target (independent antibody strategy). 

Orthogonal validation: For the antibody HMV312 specificity is suggested by the strong concordance of the immunostaining data with data from three independent RNA screening studies, including the Human Protein Atlas (HPA) RNA-seq tissue dataset, the FANTOM5 project, and the Genotype-Tissue Expression (GTEx) project, which are all summarized in the Human Protein Atlas (Tissue expression TH). TH positivity by HMV312 is very strong in the only extracranial tissues with documented TH RNA expression (adrenal gland). The additional detection of TH positivity by HMV312 in a subset of nerve fibers in various tissues is probably not reflected in previous RNA studies because the density of nerves was too low for resulting in measurable RNA quantities. The additional TH positivity in few amnion cells is also not supported by RNA data, mainly because amnion cells have not been analyzed on the RNA level. 

Comparison of antibodies: True expression of TH in all cell types (including nerves and amnion cells) found to be TH positive by HMV312 is corroborated by identical staining’s obtained by another commercially available independent antibody (termed “validation antibody”). 

Normal tissue gallery