Application
Anti-Dopamine Antibody, clone K56A detects level of Dopamine & has been published & validated for use in IH.
Research CategoryNeuroscience
Research Sub CategoryNeurotransmitters & Receptors
Immunohistochemistry: 1:500-1:2,500 using free floating sections by the PAP technique on rat dopaminergic areas.
Optimal working dilutions must be determined by end user.
PROTOCOL for Dopamine Detection by Immunohisto/cytochemistry. Example for a rat brain.
1. SOLUTIONS TO BE PREPARED - Solution must be prepared as needed.
Solution A: 0.1M cacodylate, 10g/L sodium metabisulfite, pH 6.2.
Solution B: 0.1M cacodylate, 10g/L sodium metabisulfite, 3-5% glutaraldehyde, pH 7.5.
2. RAT PERFUSION - The rat is anaesthetized with sodium pentobarbital or Nembutal and perfused intracardially through the aorta using a pump with Solution A (30 mL): 150-300 mL/min, Solution B (500 mL): 150-300 mL/min.
3. POST FIXATION: 15 to 30 minutes in Solution B, then 4 soft washes in 0.05M Tris with 8.5 g/L sodium metabisulfite, pH 7.5 (Solution C) .
4. TISSUE SECTIONING: Vibratome or cryostat sections can be used.
5. REDUCTION STEP: Sections are reduced with Solution C containing 0.1M sodium borohydride for 10 minutes. The sections are washed 4 times in solution C without sodium borohydride.
6. APPLICATION OF DOPAMINE ANTIBODY: Use a final dilution of 1:2,500-1:10,000 in Solution C containing 0.1% Triton X100 and 2% non-specific serum. Incubate 12 sections per 2 mL diluted antibody overnight, +4°C. Then wash the sections three times for 10 minutes each in Solution C. (Note that the antibody may be usable at a higher dilution. This should be explored to minimize the possibility of high background. Additionally, note that a change in the buffering system as indicated in the protocol may change the background and antibody recognition). The specific reaction is then revealed by PAP procedure.
6. SECOND ANTIBODY: Incubate the sections with a 1:50 to 1:200 dilution of goat anti-rabbit in Solution B containing 1% non-specific serum for either 3 hrs at 20°C or 2 hr at 37°C. Then wash the sections, 3 times, for 10 minutes each with Solution B.
7. PAP: Incubate the sections with the appropriate dilution of peroxidase anti-peroxidase (for free floating method) in Solution B containing 1% non-specific serum for 1-2 hours at 37°C. Then wash sections 3 times for 10 min each in solution B.
8. VISUALIZATION: The antigen-antibody complexes are visualized using DAB-4-HCl (25 mg/100 mL) (or other chromogen) in 0.05M Tris and filtrated; 0.05% hydrogen peroxide is added. Incubate the sections for 10 minutes at room temp. Stop the reaction by transferring the sections to 5 mL 0.05M Tris. Wash tissue with solution D using 2, 10 min washes. Mount sections on chrome-alum coated slides. Dry overnight at 37°C. Rehydrate sections using conventional histological procedures. Coverslip using rapid mounting media.
For research use only; not for use as a diagnostic.
Disclaimer
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
Immunogen
Dopamine-Gluteraldehyde-BSA.
Legal Information
CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany
Physical form
Purified immunoglobulin. Liquid containing 50% glycerol.
Format: Purified
Specificity
Dopamine.
The cross-reactivities were determined using an ELISA test by competition experiments with the following compounds:
Compound Cross-reactivity
Dopamine-G-BSA 1
L-DOPA-G-BSA 1/10,000
Tyrosine-G-BSA 1/36,000
Tyramine-G-BSA 1/>50,000
Noradrenaline-G-BSA 1/>50,000
Octopamine-G-BSA 1/>50,000
Adrenaline-G-BSA 1/>50,000
Dopamine 1/>50,000
Abbreviations:
(G) Glutaraldehyde
(BSA) Bovine Serum Albumin
Storage and Stability
Maintain at -20°C in undiluted aliquots for up to 6 months after date of receipt.
This product has met the following criteria to qualify for the following awards: