Anti-Tryptophan Hydroxylase/Tyrosine Hydroxylase/Phenylalanine Hydroxylase Antibody

PH8 Antibody is a murine monoclonal antibody that binds a common epitope of Tryptophan hydroxylase (TRH), Tyrosine hydroxylase (TYH) and Phenylalanine hydroxylase (PAH).1 In the literature this monoclonal antibody is cited as PH8. Tyrosine hydroxylase (TYH) is the enzyme which converts tyrosine to dihydroxyphenylalanine (L-dopa), a precursor of the catecholamine neurotransmitters dopamine, noradrenaline and adrenaline. Tryptophan hydroxylase (TRH) is the enzyme that converts 5-hydroxytryptophan to serotonin. In fresh tissue the PH8 Antibody binds to TYH and TRH so is a marker for TYH-containing catecholaminergic neurons,2 and serotonergic neurons.3,4,5 Tryptophan hydroxylase (TRH) can be used as a marker for serotonin as it converts 5-hydroxy-tryptophan to serotonin. Serotonin is rapidly metabolised and is unable to be detected by anti-serotonin antibodies in post mortem tissue. In human tissue that has been formalin-fixed, due to a change in the antigenic determinant of tyrosine hydroxylase (TYH), PH8 Antibody will bind only tryptophan hydroxylase (TRH).3,4,5 The PH8 Antibody can therefore be used to specifically identify serotonergic neurons in fixed human tissue. Phenylalanine hydroxylase (PAH) can be detected in hepatic tissue sections using the PH8 Antibody.

The PH8 Antibody can be used to identify dopaminergic and serotonergic neurons by immuno-histochemistry and for Western blot analysis, immunoprecipitation and immuno-histochemistry of TYH, PAH and TRH.

PROTOCOLS

Immunohistochemistry

The PH8 Antibody can be used for the immunohistochemical detection of dopaminergic and serotonergic neurons in human and rat brain stem tissue.

1. Tissue should be formalin fixed and stored in formalin prior to use for a minimum of five days.

2. Cryoprotect tissue using 30% sucrose in 0.1M Tris pH 7.4 buffer for 24-72 hours.

3. Cut using a sledge microtome to 50 μm thickness.

4. Wash the tissue samples using Tris buffer prior to commencing the staining procedure.

5. Treat the tissue samples for 3 x 15 minutes in 50% alcohol.

6. Treat the tissue samples for 20 minutes in 50% alcohol and 3% H2O2.

7. Treat tissue for 20 minutes with 10% normal horse serum in Tris buffer. This acts to block endogeneous H2O2 staining.

8. Dilute the PH8 Antibody in Tris buffer, add to the tissue samples and incubate for 1-3 days. Recommended dilutions are 1:2,000-1:10,000. At high antibody concentration all monoaminergic neurons are stained with no distinction between serotonegic and catecholominergic cells. By diluting the antibody concentrate, cell types are distinguishable due to variation in staining intensity. At lower concentrations (1:5,000-1:10,000 dilution) only serotonergic cells will stain.

9. Wash tissue (3 x 15 minutes), then add a biotinylated anti-mouse secondary antibody and incubate on an orbital shaker at room temperature (RT) for 1 hour.

10. Wash tissue (3 x 15 minutes) and incubate on an orbital shaker at RT for 1 hour with the tertiary complex (ELITE KIT, Vector, USA).

11. Wash tissue (3 x 15 minutes) and incubate on an orbital shaker at RT for 10 minutes with Tris buffered diamino-benzidine substrate. Add 0.1% H2O2 and incubate on an orbital shaker at RT for a further 5 minutes.

12. Mount tissue onto gelatinised slides and allow to dry prior to microscopy.

This Anti-Tryptophan Hydroxylase/Tyrosine Hydroxylase/Phenylalanine Hydroxylase Antibody, clone PH8 is validated for use in IHC, IP, WB for the detection of Tryptophan Hydroxylase/Tyrosine Hydroxylase/Phenylalanine Hydroxylase.

Format: Purified

The immunoglobulin fraction has been purified by Protein G chromatography. 500?g liquid protein at 2mg/mL in phosphate buffered saline (PBS). The product is supplied sterile-filtered through a 0.22 micron filter

The PH8 Antibody is shipped liquid at ambient temperature. Liquid material is stable for at up to 6 months when stored at 2-8°C.

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany