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. The polyclonal antibody (NCL-Ki67p) labels Ki67 antigen in the granular components of the nucleolus during late G1, S, G2 and M phases. There is a strong correlation between low or high Ki67 index and low or high grade histopathology of neoplasms.


Primary Antibody

Name: Ki67 Antibody

Clone: Rabbit anti-Human

Supplier: Novocastra

Catalog Number: NCL-Ki67-P

Dilution: 1:3000 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:
Tissue Type: Tonsil, thymus, stomach, small intestine, colon, skin, oral mucosa and breast carcinoma, bone marrow, testis, and uterus.
Fixation: Formalin fixed paraffin sections
Positive Control: Spleen, Tonsil, breast carcinomas, GI tract.
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