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PCNA Antibody Staining Protocol for Immunohistochemistry
Description: The proliferating cell nuclear antigen (PCNA) is a 36 kDa molecular weight protein also known as cyclin. The protein has also been identified as the polymerase-associated protein and is synthesized in early G1 and S phases of the cell cycle. In early S phase, PCNA has a very granular distribution and is absent from the nucleoli. At late S phase, PCNA is prominent in the nucleoli. In cells fixed with organic solvents, PCNA is seen to be strongly associated in the nuclear regions where DNA synthesis is occurring, whereas in cells fixed with aldehydes the staining is more diffuse but intense and occurs throughout the cell cycle. This is due to the presence of two basic forms of the PCNA protein, a soluble form sensitive to organic fixation and not involved in replication, and a second form that is insoluble and is associated with ongoing DNA synthesis. PCNA is a very conserved protein present not only in animal but also in plant cells.
Primary Antibody
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Name: PCNA Antibody |
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Clone: Mouse anti-Human |
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Supplier: Chemicon |
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Catalog Number: MAB4078 |
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Dilution: 1:1500 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 min/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:
| Species Reactivity: Human |
| Fixation: Formalin fixed paraffin sections, or acetone fixed frozen sections (antigen retrieval is not needed) |
| Positive Control: Skin, colon cancer |
| 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. Hall PA, et al (1990) Proliferating cell nuclear antigen (PCNA) immunolocalization in paraffin sections: an index of cell proliferation with evidence of deregulated expression in some neoplasms. J Pathol 162:285-294.
2. Yu CCW, et al (1991) Haemangiopericytomas: the prognostic value of immunohistochemical staining with a monoclonal antibody to proliferating cell nuclear antigen (PCNA). Histopathol 19:29-33.