Tyrosine Hydroxylase
| Cat Number: | AB-10312 |
|---|---|
| Conjugate: | Unconjugated |
| Size: | 100 ul |
| Clone: | POLY |
| Concentration: | 1mg/ml |
| Host: | Rb |
| Isotype: | IgG |
| Immunogen: | Two rabbits were immunized with sodium dodecyl sulfated-denatured tyrosine hydroxylase that was purified from rat pheochromocytoma. Immune sera were subjected to ammonium sulphate fractionation to purify immunoglobulins. Antibodies to tyrosine hydroxylase were then purified by affinity chromatography on a CNBr-Sepharose column to which purified tyrosine hydroxylase was bound. This antibody preparation has been compared to numerous other rabbit anti-tyrosine hydroxylase preparations with several light microscopic immunocytochemical procedures and was found to provide, as a rule, superior immunocytochemical reactivity. Preliminary data also indicate that this antibody preparation reacts well with glutaraldehyde-fixed tissue suggesting that it will be preferred antibody for ultrastructural localization studies. The antibody has also been characterized with Western blots. |
| Reactivity: | Hu, Ms, Rt |
| Applications: | Western Blot : 1:2500 |
| Purification: | Aff. Pur. |
| Background: | Specific for the 60 k tyrosine Hydroxylase in Western Blots of rat brain lysates of PG-12 cells stimulated by Okadaic Acid. The antibody cross-reacts with all mammalian and at least some non mammalian forms of the enzyme in immunolabeling and immunocytochemical studies. |
| Form: | Liquid |
| Buffer: | Purified antibody at 1mg/mL in 50% PBS, 50% glycerol plus 5mM NaN3 |
| Storage: | Store the antibody at -20°C for long term storage and refrigerate a 2-8°C for shorter term. Avoid freeze and thaw cycles. Stock solutions are stable for a minimum of 1 year at -20°C. |
Western Blot Analysis ((~62 kDa):
PC12 lysate was resolved by electrophoresis, transferred to PVDF membrane and probed with anti-tyrosine hydroxylase
Proteins were visualized using a donkey anti-rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system.
Immunofluorescent analysis of rat brain section stained with rabbit pAb to tyrosine hydroxylase, dilution 1:10,000, in red and costained with mouse mAb to pNF-H, dilution 1:1,000 in green.
The blue is Hoechst staining of nuclear DNA. Following transcardial perfusion of rat with 4% paraformaldehyde, brain was post fixed for 24 hours, cut to 45μM, and free-floating sections were stained with the above antibodies. The TH1 antibody stains the striatal TH
expressing interneurons, while the pNF-H antibody labels axons from other neuronal cells.
Western blot analysis of tissue and cell lysates using rabbit polyclonal antibody to tyrosine hydroxylase, AB-10312 dilution 1:5,000 in green:
[1] protein standard (red), [2] rat brain caudate/putmen, [3] HeLa cell extract and [4] PC12 cells. The strong band at about 58kDa corresponds to TH protein.
Immunofluorescence: Representative image from a previous lot. Anti-Tyrosine Hydroxylase (TH) staining in mouse primary neural cultures using AB-10312 shown with an FITC fluorescent secondary (green). Fixation was 4% PFA and the primary antibody incubation was 1:1000, overnight at 4°C.
AB-10312 IHC(P) on Mouse Brain Tissues Substantia Nigra. Immunohistochemical performed by: Dr. Francesca Biagioni, lab. Neurobiology of Movement Disorders, I.R.C.C.S. INM Neuromed and Dr. Maria Teresa Calierno
I.R.C.C.S. INM Neuromed
AB-10312 IHC(P) on Mouse Brain Tissues Striatum. Immunohistochemical performed by: Dr. Francesca Biagioni, lab. Neurobiology of Movement Disorders, I.R.C.C.S. INM Neuromed and Dr. Maria Teresa Calierno
I.R.C.C.S. INM Neuromed
IMMUNOHISTOCHEMISTRY PROTOCOL (Paraffin Embedded Tissues)
Deparafinized section:
– Xilene 30’;
– ETOH 100 10’;
– ETOH 96 10’;
– ETOH 70 10’;
– H2Od 5’
-Antigen retrieval:
Acetic acid 5% in ETOH 100% at RT for 15’
Unmasking with pH 9 buffer (Microwave 10’): pH 9 buffer.
Unmasking with pH 8 buffer (Microwave 10’): pH 8 buffer
-Wash in PBS 3×10’
-Triton X100 0,1% (in PBS) 15’
– Wash in PBS 3×10’
-Hydrogen peroxide 3% 10’
– Wash in PBS 3×10’
-Normal Goat Serum (NGS) 10% in PBS 1h
-Incubation with anti-TH 1:200 in PBS+NGS 2% overnight a +4C in humid chamber
– Wash in PBS 3×10’
– Incubation with Ab II biotinilated anti-goat 1:200 in PBS 1h
– Wash in PBS 3×10’
– Horseradish peroxidase streptavidin 1:100 in PBS 1h
– Wash in PBS 3×10’
-Develop with chromogen (DAB + metal enhancer) 3’
PROCEDURE OF IMMUNOFLUORESCENT STAINING OF FREE-FLOATING BRAIN TISSUE SECTIONSTISSUE PREPARATION:
1. Perfuse transcardially the animal (rat or mouse) with ice-cold PBS (pH7.4), followed by freshly made 4% paraformaldehyde fixative solution in PBS.
2. Postfix the removed brain in the same 4% paraformaldehyde fixative solution in PBS (4°C for 16 – 24 hours).
3. Cryoprotect the tissue by immersing it in sucrose solutions in PBS (15%, for 24 hours followed by 30% until tissue will sink, may take from 48 hours up to 1 week).
4. Cut 40 – 50μm sections on a cryostat.
5. Keep sections in PBS + 0.05M NaN3 at 4°C
until they were taken for staining. During the staining process, the sections should never be allowed to dry out.