Anti-Choline Acetyltransferase Antibody

ラット脳から精製されたコリンアセチルトランスフェラーゼ。

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.

この抗コリンアセチルトランスフェラーゼ抗体、クローン1E6を用いたコリンアセチルトランスフェラーゼの検出は、IHでの使用が検証されています。

研究のカテゴリ
神経科学

研究サブカテゴリー
神経伝達物質および受容体

神経細胞およびグリアマーカー

脳および脊髄（CNS）のコリン作動性ニューロンを認識します。

未精製

防腐剤を含まない腹水。

-20°Cで出荷日から1年間保存できます。凍結融解を繰り返さないために、分注してください。製品を最大限回収するために、融解後のキャップを外す前に元のバイアルを遠心分離します。

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濃度：ロットに固有の濃度につきましては試験成績書をご参照ください。

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

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