|Immunogen:||Rat tyrosine hydroxylase(TH)|
Western blot: 1:1000
|Purification:||Immunogen Affinity Purified.|
Tyrosine hydroxylase is involved in the conversion of phenylalanine to dopamine. As the rate-limiting enzyme in the synthesis of catecholamines, tyrosine hydroxylase has a key role in the physiology of adrenergic neurons. Human TH gene contains 13 primary exons and spans approximately 8 kb. TH is in the 11p15.5 region
A section of mouse midbrain stained with mouse monoclonal antibody to tyrosine hydroxylase TH-100 in green. The cytoplasm and processes of these dopaminergic neurons are revealed.
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.
Anti-Tyrosine Hdroxylase Antibody in IHC (P) 1:100 on mouse brain tissue – (substantia nigra & striatum).
IMMUNOHISTOCHEMISTRY (Paraffin Embedded Tissues) SABC- METHOD Preparation of tissue (SABC)
• Prepare three bottles of 90%, 95% and 100% dimethylbenzene and three bottles of 90%, 95% and 100% ethanol.
• Immerse paraffin sections into three bottles of dimethylbenzene orderly (from low to high), 7min each.
Then immerse into ethanol orderly at room temperature (from high to low), 7min each. Wash with water to remove ethanol.
Note: The process of dewaxing should be done in a fume hood at room temperature in summer. When the temperature is lower than 18°C, it is recommended to dewax at 50°C.
• Mix 30% H2O2 with distilled water(1:9). Immerse dewaxed paraffin section into the 3%H2O2 at room temperature for 10mim. Wash with distilled water for several times.
• Heat repair: Immerse the paraffin sections into Citrate buffer (pH6.0), heat until boiling in the
microwave and then cut off the power, keep it in the microwave for nearly 5~10 mins. Repeat 1 or 2 times. Then cool at room temperature and wash 1 or 2 times with PBS(pH7.2-7.6)
Add 5% BSA blocking solution or Normal goat serum and incubate at 37°C for 30min. Discard extra liquid, no washing.
4) Adding primary antibody
Dilute primary antibody: 1ug/ml is regarded as the concentration. Add some antibody diluents solution to primary antibody. Incubate at 37°C for 2 hours or overnight.
5) Wash with PBS for 2 times, 20min each. Add biotin-conjugated secondary antibody and incubate at 37°C for 30min.
6) Wash with PBS for 2 times, 20min each. Add SABC reagents and incubate at 37°C for 30 min. Wash with PBS for 3 times, 20min each.
7) Add DAB reagent and incubate at RT. Wash with distilled water for several times.
8) Counterstain with haematoxylin. Dehydration and then immerse paraffin sections into dimethylbenzene twice, 7min each. Observe with microscope after seal