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Ki67 Antibody Staining Protocol for Immunohistochemistry
Description: The assessment of cell proliferation by the detection of Ki67 antigen in neoplastic cell populations has been shown to be of prognostic value.
Primary Antibody
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Name: Ki67 Antibody |
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Clone: MIB-1, Mouse anti-Human |
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Supplier: Dako |
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Catalog Number: M7240 |
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Dilution: 1:100 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 as well as cell smears. |
| Positive Control: Tonsil, thymus, stomach, small intestine, colon, skin, oral mucosa and breast carcinoma, bone marrow, testis, and uterus. |
| 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. Charafe-Jauffret E, et al (2004) Immunophenotypic analysis of inflammatory breast cancers: identification of an inflammatory signature. J Pathology. 202:265-273.
2. Scott IS, et al (2003) A novel immunohistochemical method to estimate cell-cycle phase distribution in archival tissue: implications for the prediction of outcome in colorectal cancer. J Pathology 201:187-197.
3. Heuschmid M, Hofmockel G, Dexler B, Dammrich J, Bassukas ID. (2002) Different antigen unmasking techniques lead to significant differences in immunohistochemical staining of Ki-67 (Mib-1) in renal cell carcinomas. Oncol Rep. Jan-Feb;9(1):19-22. PubMed Abstract
4. Boon ME (1996) Microwave-antigen retrieval: the importance of pH of the retrieval solution for MIB-1 staining Eur J Morphol. 34(5):375-9. PubMed Abstract