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

Name: Ki67 Antibody

Clone: MIB-1, Mouse anti-Human

Supplier: Dako

Catalog Number: M7240

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.

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

Reagent: Mayer's Hematoxylin
Staining Time: 30 seconds

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



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