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Ki67 can be a challenging target to work with in some applications.
Expression of Ki67 occurs preferentially during late G1, S, G2 and M phases of the cell cycle, while in cells in G0 phase the antigen cannot be detected (at protein level).6
|Sample fixation||The ideal fixation time will depend on the size of the tissue block and the type of tissue, but fixation between 18–24h is suitable for most samples.|
|Under-fixation can lead to edge staining, with strong signal on the edges of the section and no signal in the middle.|
|Over-fixation can mask the epitope; antigen retrieval can help overcome this masking, but if the tissue has been fixed for a long period of time (i.e. over a weekend), there may be no signal even after antigen retrieval.|
|Maximizing signal||Antigen retrieval: Heat in citrate buffer pH 6 for 20-30 minutes or enzymatic (trypsin, proteinase K). (Necessary if fixed in PFA)|
|Permeabilize the tissues: 0.2% Triton in PBS for 10 minutes|
Human tonsil tissue
Mouse tumour tissue
Mouse embryonic skin tissue
Rat oesophagus, small intestine and liver tissue
Ki67 locates in chromosomes and nucleus. Therefore, 4% PFA fixation is recommended. And permeabilizing the cells (0.1% TritonX-100 in PBS for 5 minutes) in ICC assays is essential.
|Sample fixation||For nuclear proteins, fix cells in 4% PFA (20 minutes, room temperature) is recommended.|
|Do not over-fix your samples, as this will reduce signal.|
|Permeabilization||It is recommended to incubate cells with 0.1% Triton-X for 5 min to detect nuclear antigen.|
HeLa and HAP1 cells
Find full information on working with Ki67: