MAB305
Anti-Choline Acetyltransferase Antibody, clone 1E6
ascites fluid, clone 1E6, Chemicon®
Synonyme(s) :
ChAT, Choline Acetylase, CHOACTase
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About This Item
Code UNSPSC :
12352203
eCl@ss :
32160702
Nomenclature NACRES :
NA.41
Produits recommandés
Source biologique
mouse
Niveau de qualité
Forme d'anticorps
ascites fluid
Type de produit anticorps
primary antibodies
Clone
1E6, monoclonal
Espèces réactives
human, rat, monkey
Fabricant/nom de marque
Chemicon®
Technique(s)
immunohistochemistry: suitable
Isotype
IgG1
Numéro d'accès NCBI
Numéro d'accès UniProt
Conditions d'expédition
dry ice
Modification post-traductionnelle de la cible
unmodified
Informations sur le gène
human ... CHAT(1103)
rat ... Chat(290567)
rhesus monkey ... Chat(709977)
Spécificité
Recognizes cholinergic neurons in the brain and spinal cord (CNS).
Immunogène
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.
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
Neuroscience
Research Sub Category
Neurotransmitters & Receptors
Neuronal & Glial Markers
Neurotransmitters & Receptors
Neuronal & Glial Markers
Forme physique
Ascites fluid containing no preservatives.
Unpurified
Stockage et stabilité
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.
Remarque sur l'analyse
Control
Brain tissue
Brain tissue
Autres remarques
Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.
Informations légales
CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany
Clause de non-responsabilité
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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Code de la classe de stockage
10 - Combustible liquids
Classe de danger pour l'eau (WGK)
WGK 1
Point d'éclair (°F)
Not applicable
Point d'éclair (°C)
Not applicable
Certificats d'analyse (COA)
Recherchez un Certificats d'analyse (COA) en saisissant le numéro de lot du produit. Les numéros de lot figurent sur l'étiquette du produit après les mots "Lot" ou "Batch".
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