Acetylcholinesterase Antibody Staining Protocol for Immunohistochemistry

Description: Acetylcholinesterase rapidly hydrolyses acetylcholine to acetate and choline thereby inactivating it. Acetylcholinesterase (AChE) is found in the neuromuscular junction anchored to the basal lamina which runs between the nerve terminal and muscle membrane. The involvement of AChE-rich cholinergic neurones is of interest in research studies of human dementias. AChE is also found outside the nervous and neuromuscular systems in blood, lymph, germ and liver cells which may suggest another role for AChE, not related to cholinergic transmission.

Primary Antibody

Name: Acetylcholinesterase

Clone: HR2, Mouse Anti-Human

Supplier: Novocastra Labs

Catalog Number: NCL-AChE

Dilution: 1:20 - 1:40 using IHC-Tek^TM Antibody Diluent (Cat# IW-1000 or IW-1001) to reduce background and unspecific staining and serum blocking step is NOT needed.

Incubation Time/Temp: 60 min/room temperature

Antigen Retrieval

Device: N/A

Buffer/pH value: N/A

Heat/Cool Temperature: N/A

Heat/Cool Time: N/A

Detection Methods

Standard Method: ABC Method or LSAB Method

Enhanced Method: Polymeric Methods

Chromogen Substrate

Reagent: DAB

Incubation Time/Temperature: 1-3 minutes/room temperature

Counterstain

Reagent: Mayer's Hematoxylin

Staining Time: 30 seconds

Results:

Staining Pattern: Cytoplasmic

Images: Search image

Additional Information:

Tissue Type: Human cerebellum

Fixation: Acetone fixed frozen sections

Positive Control: Brain

Negative Control: Omit primary antibody, isotype control, absorption control

Blocking: 2-5% normal serum to reduce unspecific background staining; 0.5-3% H2O2 to block endogenous peroxidase activity; avidin/biotin to block endogenous biotin activity if necessary

References:

1. Andra J, Lachmann I, Luppa H. (1988) A comparison of the localization of acetylcholinesterase in the rat brain as demonstrated by enzyme histochemistry and immunohistochemistry. Histochemistry. 88(3-6):595-601. PubMed Abstract.

2. Mizukawa K, McGeer PL, Tago H, Peng JH, McGeer EG, Kimura H. (1986) The cholinergic system of the human hindbrain studied by choline acetyltransferase immunohistochemistry and acetylcholinesterase histochemistry. Brain Res. 1986 Jul 30;379(1):39-55. PubMed Abstract.

3. Mesulam MM, Mufson EJ, Levey AI, Wainer BH.Atlas of cholinergic neurons in the forebrain and upper brainstem of the macaque based on monoclonal choline acetyltransferase immunohistochemistry and acetylcholinesterase histochemistry. Neuroscience. 1984 Jul;12(3):669-86. PubMed Abstract.