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Product details

Synonyms = tyrosine hydroxylase , DYT14 , DYT5b , TYH

Antibody type = Recombinant Rabbit monoclonal / IgG

Clone = HMV-312

Positive controlAdrenal 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.

Cellular localization = Nucleus

Reactivity = Human

 

 

Application = Immunohistochemistry
Dilution = 1:100 – 1:200
Intended Use = Research Use Only

Relevance of Antibody

Tyrosine Hydroxylase is a Rate limiting enzyme for catecholin synthesis.

Biology Behind

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.

Staining Pattern in Normal Tissues

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 HMV-312 are shown in our “Normal Tissue Gallery”.

Brain Cerebrum Negative.
Cerebellum Negative.
Endocrine Tissues Thyroid Negative.
Parathyroid Negative.
Adrenal gland Strong membranous and cytoplasmic TH staining of medullary cells. Some nerve fibers in the cortex also show distinct TH positivity.
Pituitary gland Negative.
Respiratory system Respiratory epithelium Negative.
Lung Negative.
Gastrointestinal Tract Salivary glands Negative. Some nerve fibers exhibit distinct TH positivity.
Esophagus Negative.
Stomach Negative.
Duodenum Negative.
Small intestine Negative.
Appendix Negative. Distinct TH staining of few nerve fibers in the muscular wall.
Colon Negative.
Rectum Negative.
Liver Negative.
Gallbladder Negative.
Pancreas Negative.
Genitourinary Kidney Negative. Distinct TH staining of few nerve fibers in the muscular wall of the kidney pelvis.
Urothelium Negative.
Male genital Prostate Negative. Many nerve fibers exhibit distinct TH positivity.
Seminal vesicles Negative. Many nerve fibers exhibit distinct TH positivity.
Testis Negative.
Epididymis Negative.
Female genital Breast Negative.
Uterus, myometrium Negative.
Uterus, ectocervix Negative.
Uterus endocervix Negative. Some nerve fibers exhibit distinct TH positivity.
Uterus, endometrium Negative.
Fallopian Tube Negative.
Ovary Negative. Some small nerves show distinct TH positivity of nerve fibers. Individual nerve fibers also show TH positivity.
Placenta early Negative.
Placenta mature Negative.
Amnion Distinct cytoplasmic and membranous TH positivity of a small subset of amnion cells.
Chorion Negative.
Skin Epidermis Negative.
Sebaceous glands Negative.
Muscle/connective tissue Heart muscle Negative. Strong TH staining of fibers in some nerves.
Skeletal muscle Negative.
Smooth muscle Negative. Strong TH staining of some nerve fibers in the muscular wall of various organs. The density of TH positive nerve fibers may variy dependent on the organ and the location within the organ.
Vessel walls Negative.
Fat Negative.
Stroma Negative.
Endothelium Negative.
Bone marrow/lymphoid tissue Bone marrow Negative.
Lymph node Negative.
Spleen Negative.
Thymus Negative.
Tonsil Negative.
Remarks

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

 

Positive controlAdrenal 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

Staining Pattern in Relevant Tumor Types

TH expression occurs in pheochromocytomas (almost always positive) and paraganglioma (about 30% positive) and rarely in other tumors.

The TCGA findings on TH RNA expression in different tumor categories have been summarized in the Human Protein Atlas.

 

 

Cancer tissue gallery

Compatibility of Antibodies

No data available at the moment

Protocol Recommendations

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 HMV-312 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.

Potential Research Applications

  • 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.

Evidence for Antibody Specificity in IHC

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 HMV-312 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 HMV-312 is very strong in the only extracranial tissues with documented TH RNA expression (adrenal gland). The additional detection of TH positivity by HMV-312 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 HMV-312 is corroborated by identical staining’s obtained by another commercially available independent antibody (termed “validation antibody”). 

 

Normal tissue gallery