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CNPase Antibody Staining Protocol for Immunohistochemistry
Description: CNPase (2', 3'-cyclic nucleotide 3'-phosphodiesterase [or -phosphohydrolase], EC 3.1.4.37) 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
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Name: CNPase Antibody |
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Clone: 11-5B, Mouse monoclonal |
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Supplier: Chemicon |
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Catalog Number: MAB326 |
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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. |
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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 |
Counterstain
| Reagent: Mayer's Hematoxylin |
| Staining Time: 30 seconds |
Results:
| 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 |
Notes:
Not tested on frozen sections |
References: