Acetylcholine (ACH) antibody

Antigen: Acetylcholine (ACH)

Clonality: Polyclonal

Host: Rabbit

Application: Immunohistochemistry (IHC)

Catalog no.: ABIN94523

Additional Information

Alternative name: Acetylcholine

Immunogen: Synthetic choline-glutaric acid conjugated to protein carrier

Format: Lyophilized

Isotype: IgG  (Matching secondary antibodies)

Specificity: Conjugated choline-glutaric acid . Using a conjugate choline-glutaric acid-Protein, antibody specificity was performed with an ELISA test by competition experiments with the following compounds: Compounds Cross-reactivity ratio (a) Choline-GA-PL 1 Choline-allyl alcohol-G-BSA 1/1.6 Phosphatidylcholine 1/>10,000 Choline 1/>10,000 Acetylcholine 1/>10,000 (a): Choline-GA-BSA concentration /unconjugated or conjugated analogs concentration at half displacement. GA = Glutaric anhydride PL = Poly lysine BSA = Bovine Serum Albumin

Application Details

Application Notes: Example of Immunocytochemical applications Detection of conjugated choline-glutaric acid in rat brain 1- Perfusion: The rat is anesthetized with sodium Pentobarbital or Nembutal and perfused intracardially through the aorta using a pump with the following fixative solution: glutaraldehyde 0.5M, 2-nitrobenzyl alcohol 0.1M, sodium metabisulfite 2- Post fixation: 2h in 0.5M glutaraldehyde solution (pH 7.5) without the 2-nitrobenzyl alcohol, then 4 soft washes in Tris 0.05M with sodium metabisulfite 10g/l, pH 7.4 (solution B). 3- Tissue sectionning: Cryostat or vibratome sections can be used. The sections were washed 4 times in solution B, and incubated for 1h at 37°C in solution B containing 0.2% triton X100, plus 1% of non specific serum. 4- Application of anti-conjugated Acetylcholine antibody: The final dilution is 1/1000 to 1/5,000 in solution B containing 0,2% triton X100, plus 1% 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 B. N.B.: The antibody may be used at a higher dilution. The customer should explore the further 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. 5- PAP procedure: Second antibody: Sections are incubated with 1/200 dilution of goat anti-rabbit antibodies diluted at 1/100 in solution B for 3 hours at 20°C or 1 hour at 37°C. Then, they are washed 3 times (10 min) with solution B PAP: Sections are incubated with mouse peroxidase/anti-peroxidase complex diluted at 1/500 in solution B for 1 hour at 37°C. Then, they are washed 3 times (10 min) with solution B Revelation: Antibody-antigen complexes are revealed using diaminobenzidine (25mg/100ml) (or other chromogen) dissolved in Tris 0.05M and filtrated 0.3% of nickel ammonium sulfate 0.05% of H2O2 were added. The sections are incubated for 10 min at 20°C. Reaction is stopped by transfering sections in 5ml of Tris 0.05M. Recommended dilutions for Immunocytochemistry (1/1000-1/5,000) Recommended dilutions for Western Blot (1/1,000-1/2,000)

Purity: Antiserum previously preabsobed on protein carriers, and purified

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

Research Area: Neurotransmitters

Restrictions: For Research Use only

Publications

Publications: Wang-Bennett, Sovan, Glantz: "Immunocytochemical studies of the distribution of acetylcholine in the crayfish brain." in: The Journal of comparative neurology, Vol. 273, Issue 3, pp. 330-43, 1989 (PubMed).

McRae-Degueurce, Geffard: "One perfusion mixture for immunocytochemical detection of noradrenaline, dopamine, serotonin and acetylcholine in the same rat brain." in: Brain research, Vol. 376, Issue 1, pp. 217-9, 1986 (PubMed).

Geffard, McRae-Degueurce, Souan: "Immunocytochemical detection of acetylcholine in the rat central nervous system." in: Science (New York, N.Y.), Vol. 229, Issue 4708, pp. 77-9, 1985 (PubMed).

Geffard, Vieillemaringe, Heinrich-Rock et al.: "Anti-acetylcholine antibodies and first immunocytochemical application in insect brain." in: Neuroscience letters, Vol. 57, Issue 1, pp. 1-6, 1985 (PubMed).