Specificity

Recognizes cholinergic neurons in the brain and spinal cord (CNS).

Immunogen

Choline acetyltransferase purified from rat brain.

Application

Detect Choline Acetyltransferase using this Anti-Choline Acetyltransferase Antibody, clone 1E6 validated for use in IH.

Immunohistochemistry: 1:100-1:250. See immunohistochmistry procedure below.

Optimal working dilutions must be determined by the end user.

IMMUNOHISTOCHEMISTRY PROCEDURE (PAP TECHNIQUE) FOR MAB305, MONOCLONAL ANTIBODY TO CHOLINE ACETYLTRANSFERASE

I) Perfusion & Sectioning Procedure

1. Perfuse through the heart with a fixative solution containing 4% paraformaldehyde in 0.12 M phosphate buffer (pH 7.3) for light microscopy (LM), and additionally, 0.1% gluteraldehyde and .002% CaCl2 for electron microscopy (EM).

2. Remove brain and postfix 2-18 hours at 4°C in 4% paraformaldehyde in 0.12 M phosphate buffer.

3. After brain is blocked for sectioning, wash in several changes of buffer for 2-3 hours.

4. Specimens for EM are sectioned on a Vibratome (50 μm) and rinsed in buffer, those for LM should be cryoprotected in 30% sucrose in buffer.

5. After freezing with dry ice, 30-40 μm thick sections of LM specimens are cut on a cryostat.

6. Sections are rinsed, and then stored in phosphate buffer containing 0.1% sodium azide.

II) Staining Procedure

Tissue is processed as freely-floating sections in continuously agitated solutions. All incubations are performed at room temperature unless otherwise stated.

1.a. For localizing ChAT-positive somata and dendrites:

Sections are washed in 0.1 M Tris-buffered saline (TBS; containing 1.4% NaCl, pH 7.3) only. No detergent or enzyme pretreatment is used.

b. For localizing ChAT-positive terminal-like structures:

Incubate sections in TBS (pH 8.1) for 5 minutes at 37°C. Transfer sections to TBS (pH 8.1) containing pronase (1.2 μg/mL) for 1 1/2-2 minutes at 37°C, followed by several ice cold buffer washes for a total of 5 minutes. The concentration of pronase and incubation time of the digestion should be evaluated for each region examined.

c. For localizing ChAT immunoreactivity and subsequently counterstaining the sections:

Incubation in TBS containing 0.1%-0.8% Triton X-100 for 15 minutes may increase the tissue penetration of the immunoreagents, but it also raises the background staining.

2. Incubate sections in normal goat serum (3-5%) for one hour. The working solutions of all antisera should also contain similarly diluted normal goat serum.

3. Incubate in anti-ChAT monoclonal antibody solution (Suggested working dilution 1:250, final working dilution must be determined by end user) for 2 hours at room temperature and then for an additional 6-18 hours at 4°C.

4. Incubate with second antibody (i.e. Goat anti-Mouse IgG, Cat. No.: AP124, dilution 1:50-100) for 1-2 hours.

5. Incubate with diluted PAP complex (i.e. Mouse PAP, Cat No.: PAP14, conc. 25-50 μg/mL) for one hour.

6. After rinsing in buffer, the second antibody and PAP steps are repeated for 40 minutes to 1 hour each in order to amplify staining intensity, particularly of small ChAT-containing structures.

7. React for 15 minutes with 0.06% 3,3′-diaminobenzidine×4 HCl (DAB; diluted in phosphate buffered saline, pH 7.3) and 0.006% H2O2.

8. Specimens for routine LM are postfixed for 1 minutes in 0.005% OsO4 (osmium tetraoxide), and then mounted, dehydrated and coverslipped. Selected regions blocked for EM are postfixed in 2% OsO4 for 1 hour, en bloc stained with uranyl acetate, and flat-embedded in Epon-Araldite resin.

Research Category
Neuroscience

Research Sub Category
Neurotransmitters & Receptors

Neuronal & Glial Markers

Physical form

Ascites fluid containing no preservatives.

Unpurified

Storage and Stability

Maintain for 1 year at -20°C from date of shipment. Aliquot to avoid repeated freezing and thawing. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.

Analysis Note

Control
Brain tissue

Other Notes

Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.

Legal Information

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

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.

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