CNPase Antibody Staining Protocol for Immunohistochemistry


Description: CNPase (2', 3'-cyclic nucleotide 3'-phosphodiesterase [or -phosphohydrolase], EC is present in very high levels in brain and peripheral nerve. This enzyme is found almost exclusively in oligo-dendrocytes and Schwann cells, the cells that form myelin in the central and peripheral nervous system, respectively. Therefore, CNPase has been very useful as a marker for these particular cell types. Developmentally, this enzyme appears early in oligodendrocytes, earlier than most other myelin proteins. It continues to be expressed at high levels in oligodendrocytes and Schwann cells of adult animals. In various diseases, neurological mutants, and in experimental conditions in which myelin is reduced, CNPase levels may be severely reduced. The enzyme itself exists in two forms in most species (three in mouse) with molecular masses of 48 and 46 kD on SDS-PAGE, referred to as CNP2 and CNP1, respectively.Studies continue to elucidate the physiological role of the enzyme.


Immunohistochemical localization of CNPase has shown the enzyme to be restricted to oligodendrocytes and Schwann cells. The enzyme appears to be distributed in single and double loose wraps of myelin and not in compact myelin as earlier thought by most investigators. CNPase is located on the inner and outer loops of myelin, paranodally and near the inner surface of the oligodendrocyte membrane. In plaque regions of multiple sclerosis patients, the enzyme is reduced, and when CNS myelin is decreased, CNPase is one of the earlier proteins to be lost from the myelin. In addition, an enzyme that is probably identical to brain CNPase is located in the outer rod segments within the visual system, and this protein is also recognized by the monoclonal antibody 11-5B. In mixed human gliomas, the enzyme levels are reduced, although about 5% of the oligodendrocytes occasionally show normal positive staining. Both frozen and paraffin sections.


Primary Antibody

Name: CNPase Antibody

Clone: 11-5B, Mouse monoclonal

Supplier: Chemicon

Catalog Number: MAB326

Dilution: 1:200 using IHC-TekTM 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 minutes/room temperature

Antigen Retrieval
Device: IHC-TekTM Epitope Retrieval Steamer Set (Cat# IW-1102)
Buffer/pH value: IHC-TekTM Epitope Retrieval Solution (Cat# IW-1100)
Heat/Cool Temperature: 95-100 ºC/room temperature
Heat/Cool Time: 20 minutes/20 minutes

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

Reagent: Mayer's Hematoxylin
Staining Time: 30 seconds

Staining Pattern: Nuclear
Images: Search image

Additional Information:
Tissue Type: Brain (glia tumor)
Fixation: Formalin fixed paraffin sections
Positive Control: Brain tumor
Negative Control: Omit primary antibody, isotype control or 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


Not tested on frozen sections