Anti-Acetylcholine Antibody

Acetylcholine-Glutaric anhydride-PL

Anti-Acetylcholine Antibody is an antibody against Acetylcholine for use in IH.

Immunohistochemistry: 1:250-1:2500 by PAP (see suggested protocol below).

Optimal working dilutions must be determined by the end user.

PROTOCOL for Acetylcholine Detection by Immunohisto/cytochemistry. Example for a rat brain.

1. SOLUTIONS TO BE PREPARED - Solution must be prepared as needed.

Solution A: 0.5M glutaraldehyde, 0.1M 2-nitrobenzyl alcohol, 10g/L sodium metabisulfite, 0.1 M cacodylate, 0.01M sodium bromide, pH 10.7.

Sometimes a precipitate may form while making solution A. To avoid this, first prepare a mixture with 1.0M glutaraldehyde and 0.2M 2-nitrobenzyl alcohol. Prepare a second mixture with 20g/L sodium metabisulfite and 0.2M cacodylate; add sodium bromide to obtain pH 10.7. Then combine the two mixtures in equal parts while stirring.



Solution B: 0.05 M Tris, 10.0 g/L sodium metabisulfite, pH 7.4.(*)

2. RAT PERFUSION - The rat is anaesthetized with sodium pentobarbital or Nembutal and perfused intracardially through the aorta using a pump with Solution A (200-300 mL/min).

3. POST FIXATION: 2 hours in 0.5M glutaraldehyde solution, (pH 7.5) without the 2-nitrobenzyl alcohol, then 4 soft washes in solution B.

4. TISSUE SECTIONING: Vibratome or cryostat sections can be used.

The sections are washed 4 times in solution B, and incubated for 1 hour at 37°C in solution B containing 0.2% Triton X100, plus 1% non specific serum.

5. APPLICATION OF ACETYLCHOLINE ANTIBODY: Use a final dilution of 1:500-1:2,500 in Solution B containing 0.2% Triton X100 and 1% 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 B. (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.

Research Category
Neuroscience

Research Sub Category
Neurotransmitters & Receptors

Acetylcholine



The cross-reactivities were determined using either ELISA or RIA techniques, at concentration/unconjugated or conjugated amino acid concentration at half displacement with the following compounds:



Compound Cross-reactivity

Choline-GA-PL 1

Phosphatidylcholine 1/>10,000

Choline 1/>10,000

Acetylcholine 1/>10,000

Choline-allyl alcohol-G-BSA 1/1.6



The antisera was also tested for specificity using the free-floating PAP technique on rat cortex.



Abbreviations:



(GA) Glutaric anhydride

(=) Non-reduced conjugate

(PL) Poly lysine

(BSA) Bovine Serum Albumin

Rabbit antiserum. Liquid with 0.05% sodium azide.

Maintain at -20°C in undiluted aliquots for up to 6 months after date of receipt. Avoid repeated freeze/thaw cycles.



During shipment, small volumes of antibody will occasionally become entrapped in the seal of the product vial. For antibodies with volumes of 200 μl or less, we recommend gently tapping the vial on a hard surface or briefly centrifuging the vial in a tabletop centrifuge to dislodge any liquid in the container′s cap.

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

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.