Ki67 Antibody Staining Protocol for Immunohistochemistry


Description: The Ki-67 antigen is a large nuclear protein preferentially expressed during all active phases of the cell cycle (G1, S, G2 and M phases), but absent in resting cells (G0) cells. Antibodies against the Ki-67 antigen have proven valuable by allowing direct monitoring of the growth fraction of a pool of cells. The TEC-3 antibody is the antibody of choice for the demonstration of the Ki-67 antigen in formalin-fixed, paraffin-embedded mouse specimens.


Primary Antibody

Name: Ki67 Antibody

Clone: TEC-3, Rat anti-Mouse

Supplier: Dako

Catalog Number: M7249

Dilution: 1:50 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
Positive Control: Thymus, GI tract, skin, breast carcinoma.
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