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

Name: Ki67 Antibody

Clone: MIB-5, Mouse anti-Rat

Supplier: Dako

Catalog Number: M7248

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: 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



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.