Dihydroxyphenylalanine, L- Antikörper

Antigen: Dihydroxyphenylalanine, L-

Klonalität: Polyklonal

Wirt: Kaninchen

Applikation: Immunzytochemie (ICC)

Produktnummer: ABIN98430

Produktbeschreibung

Immunogen: Synthetic L-DOPA conjugated to protein carrier (Pc)

Format: Lyophilized

Isotyp: IgG  (Passende Sekundärantikörper)

Spezifität: Conjugated L-dihydroxyphenylalanine (L-DOPA) . Using a conjugate L-DOPA-(Pc), antibody specificity was performed with an ELISA test by competition experiments with the following compounds: Compounds Cross-reactivity ratio (a) L-DOPA-G-(Pc) 1 -methyl-L-DOPA-G-(Pc) 1/2,200 3-O-methyl-L-DOPA-G-(Pc) 1/>50,000 Dopamine-G-(Pc) 1/>50,000 Noradrenaline-G-(Pc) 1/>50,000 Tyrosine-G-(Pc) 1/>50,000 (a): L-DOPA-G-(Pc) concentration /other conjugated catecholamine concentration at half displacement G = Glutaraldehyde

Anwendungen

Applikationshinweise: Example of cytochemistry application Detection of conjugated L-DOPA in rat brain 1- Perfusion: The rat is anaesthetized with sodium Pentobarbital or Nembutal and perfused intracardially through the aorta using a pump with the following solutions: Solution A: cacodylate 0.1M, sodium metabisulfite 10g/l, pH = 6.2 Solution B: cacodylate 0.1M, sodium metabisulfite 10g/l and glutaraldehyde 3-5%, pH = 7.5 2- Post fixation: 15 to 30 min in solution B, then 4 soft washes in Tris 0.05M with sodium metabisulfite 8.5g/l, pH 7.5 (solution C). 3- Tissue sectionning: Cryostat or vibratome sections can be used. 4- Reduction step: Sections are reduced with the solution C containing sodium borohydride (0.1M) for 10 min. Then, the sections are washed 4 times with solution C without sodium borohydride. 5- Application of anti-conjugated L-DOPA antibodies: The final dilution is 1/1,000 to 1/5,000 in solution C containing triton X100 0.5%, plus 2% of non-specific serum. A dozen of sections can be incubated with 2ml of antibody solution overnight at 4°C. Then, after this period, the sections are washed 3 times (10 min) with solution C. N.B.: Antibodies may be used at a higher dilution. The customer should explore the antibody dilution to reduce the possibility of high background. Note that a substitution in the buffer system as used in our protocol may change the background and the antibody recognition. 6- PAP procedure: Second antibody: Sections are incubated with 1/100 dilution of goat anti-rabbit in solution C for 3 hours at 20°C or 1 hour at 37°C. Then, they are washed 3 times (10 min) with solution C PAP: Sections are incubated with 1/1,000 dilution of rabbit peroxidase anti-peroxidase complex in solution C for 1 hour at 37°C. Then, they are washed 3 times (10 min) with solution C Revelation: Antibody-antigen complexes are revealed using diaminobenzidine (25mg/100ml) (or other chromogen) dissolved in Tris 0.05M and filtrated 0.05% of H 2 O 2 is added. The sections are incubated for 10 min at 20°C. Reaction is stopped by transfering sections in 5ml of Tris 0.05M. Gemacbio sells the same and other antibodies to conjugated small molecules raised in mouse:used together, these tools could be helpful for immunocytochemistry double labelling. Double detection of conjugated L-DOPA and Dopamine in rat brain 1- Perfusion: The rat will be deeply anesthetized with sodium Pentobarbital or Nembutal and perfused intracardially through the aorta using a pump with the 500ml of 5% glutaraldehyde (G), 0.9% sodium metabisulfite (SMB) solution in 0.1cacodylate buffer pH 7,4. 2- Post fixation: 2h, 4°C in the same fixative solution. 3-Tissue sectionning: Cryostat or vibratome sections can be used. 4- Application of anti-conjugated antiserum: Sections will be reduced in 0.05M Tris buffer containing 0.9% SMB (Tris- SMB). Then, the sections will be washed in the same solution (12h, 4°C) and incubated in Tris-SMB containing 3% non specific serum and 0.1% Triton X100 (8h at 4°C). 5- Application of anti-conjugated L-DOPA antibodies: Free floating adjacent sections will be incubated (24h, 4°C) with a polyclonal antiserum against conjugated L-DOPA (1/,1000 to 1/5,000), with a monoclonal antibody against conjugated DA (1/,1000 to 1/5,000), and with both. Antisera will be diluted in Tris-SMB, 1% non-specific serum, 0.2% Triton X100 solution. N.B.: Antibodies may be used at a higher dilution. The customer should explore the antibody dilution to reduce the possibility of high background. Note that a substitution in the buffer system as used in our protocol may change the background and the antibody recognition. Recommended dilutions for Immunocytochemistry (1/1,000-1/5,000) Recommended dilutions for Western Blot (1/1,000-1/2,000)

Reinheit: Antiserum previously preabsorbed on protein carriers, and purified

Lagerung: Lyophilized antibodies are stable at least 2 years. After reconstitution with 50µl of distilled water and 50µl of glycerol, the aliquot can be repeated freezed (up to five times).

Beschränkungen: Nur für Forschungszwecke einsetzbar

Publikationen

Publikationen: Lagier, Charrier, Geffard et al.: "Effects of polyclonal antibodies against conjugated L-dopa or against conjugated acetylcholine on experimental venous thrombosis." in: Thrombosis research, Vol. 65, Issue 2, pp. 275-80, 1992 (PubMed).

Manier, Feuerstein, Passagia et al.: "Evidence for the existence of L-dopa- and dopamine-immunoreactive nerve cell bodies in the caudal part of the dorsal motor nucleus of the vagus nerve." in: Journal of chemical neuroanatomy, Vol. 3, Issue 3, pp. 193-205, 1990 (PubMed).

Kitahama, Geffard, Okamura et al.: "Dopamine- and dopa-immunoreactive neurons in the cat forebrain with reference to tyrosine hydroxylase-immunohistochemistry." in: Brain research, Vol. 518, Issue 1-2, pp. 83-94, 1990 (PubMed).

Tison, Mons, Geffard et al.: "The metabolism of exogenous L-dopa in the brain: an immunohistochemical study of its conversion to dopamine in non-catecholaminergic cells of the rat brain." in: Journal of neural transmission. Parkinson's disease and dementia section, Vol. 3, Issue 1, pp. 27-39, 1991 (PubMed).

Mons, Tison, Geffard: "Existence of L-dopa immunoreactive neurons in the rat preoptic area and anterior hypothalamus." in: Neuroendocrinology, Vol. 51, Issue 4, pp. 425-8, 1990 (PubMed).

Tison, Mons, Geffard et al.: "Immunohistochemistry of endogenous L-DOPA in the rat posterior hypothalamus." in: Histochemistry, Vol. 93, Issue 6, pp. 655-60, 1990 (PubMed).

Mons, Tison, Geffard: "Identification of L-dopa-dopamine and L-dopa cell bodies in the rat mesencephalic dopaminergic cell systems." in: Synapse (New York, N.Y.), Vol. 4, Issue 2, pp. 99-105, 1989 (PubMed).

Tison, Mons, Rouet-Karama et al.: "Endogenous L-dopa in the rat dorsal vagal complex: an immunocytochemical study by light and electron microscopy." in: Brain research, Vol. 497, Issue 2, pp. 260-70, 1990 (PubMed).

Mons, Danel, Geffard: "Visualization of L-dihydroxyphenylalanine in rat brain by using specific antibodies." in: Brain research, Vol. 451, Issue 1-2, pp. 403-7, 1989 (PubMed).

Okamura, Kitahama, Mons et al.: "L-dopa-immunoreactive neurons in the rat hypothalamic tuberal region." in: Neuroscience letters, Vol. 95, Issue 1-3, pp. 42-6, 1989 (PubMed).

Kitahama, Mons, Okamura et al.: "Endogenous L-dopa, its immunoreactivity in neurons of midbrain and its projection fields in the cat." in: Neuroscience letters, Vol. 95, Issue 1-3, pp. 47-52, 1989 (PubMed).

Arai, Karasawa, Geffard et al.: "Immunohistochemical evidence that central serotonin neurons produce dopamine from exogenous L-DOPA in the rat, with reference to the involvement of aromatic L-amino acid decarboxylase." in: Brain research, Vol. 667, Issue 2, pp. 295-9, 1995 (PubMed).

Yue, Okamura, Goshima et al.: "Baroreceptor-aortic nerve-mediated release of endogenous L-3,4-dihydroxyphenylalanine and its tonic depressor function in the nucleus tractus solitarii of rats." in: Neuroscience, Vol. 62, Issue 1, pp. 145-61, 1995 (PubMed).

Nguyen-Legros, Krieger, Simon: "Immunohistochemical localization of L-dopa and aromatic L-amino acid-decarboxylase in the rat retina." in: Investigative ophthalmology & visual science, Vol. 35, Issue 7, pp. 2906-15, 1994 (PubMed).

Arai, Karasawa, Geffard et al.: "L-DOPA is converted to dopamine in serotonergic fibers of the striatum of the rat: a double-labeling immunofluorescence study." in: Neuroscience letters, Vol. 195, Issue 3, pp. 195-8, 1996 (PubMed).