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Ki67 Antibody Staining Protocol for Immunohistochemistry
Description: The MIB-5 antibody is the antibody of choice for demonstration of the Ki-67 antigen in formalin-fixed, paraffin-embedded rat specimens. The antibody also labels mouse Ki-67 antigen as well as primate tissues. The Ki-67 antigen is a large nuclear protein (345, 395 kDa) preferentially expressed during all active phases of the cell cycle (G1, S, G2 and M phases), but absent in resting (G0) cells.
Primary Antibody
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Name: Ki67 Antibody |
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Clone: MIB-5, Mouse anti-Rat |
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Supplier: Dako |
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Catalog Number: M7248 |
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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: Gill's Hematoxylin or Mayer's Hematoxylin |
| Staining Time: 30 seconds |
Results:
| Staining Pattern: Nuclear |
| Images: Search image |
Additional Information:
| Species Reactivity: Mouse |
| Fixation: Formalin fixed paraffin sections or acetone-fixed frozen sections. |
| Positive Control: Thymus, stomach, small intestine, colon, skin. |
| 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. Gerlach C, Sakkab DY, Scholzen T, Dassler R, Alison MR, Gerdes J. Ki-67 expression during rat liver regeneration after partial hepatectomy. Hepatology 1997;26:573-8. PubMed Abstract.
2. Birner P, Ritzi M, Musahl C, Knippers R, Gerdes J, Voigtlander T, et al. Immunohistochemical detection of cell growth fraction in formalin-fixed and paraffin-embedded murine tissue. Am J Pathol 2001;158:1991-6.
3. Scholzen T, Gerdes J. The Ki-67 protein: from the known and the unknown [review]. J Cell Physiol 2000;182:311-22. PubMed Abstract.