Image 1: Structure of Ki67 protein.

Ki67 Introduction

Protein Function

• Ki67 is crucial for maintaining chromosome dispersion in the cytoplasm during cell division.
• Widely used in research and pathology as a tumor marker.
• Valuable for prognosis and diagnosis of various cancers.
• Ki67 index correlates with disease progression and can assess patient survival and tumor progression.
• Associated with the number of proliferating cells rather than their rate of proliferation.

Protein Expression

• Mainly expressed in proliferating cells (protein level).
• Expression 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.
• Present at highest level in G2 phase and during mitosis (at protein level).

Protein Localization

• In interphase, forms fiber-like structures in fibrillarin-deficient regions surrounding nucleoli.
• Cell nucleus.

Figure 2: ICC experimental result of Ki67 protein using Anti-Ki67 antibody (ab16667). Green: Ki67, Red: alphaTubulin, Blue:DAPI.

Isoforms & Post-translation Modifications

• Human (P46013): Long isoform: 358.7 kDa (predicted), Short isoform: 319.4 kDa (predicted)
• Mouse (E9PVX6): Isoform 1: 350.9 kDa (predicted), Isoform 2: 324.7 kDa (predicted)
• Rat (D4A0Y6): 343.4 kDa (predicted)
• Phosphorylated or highly phosphorylated in mitosis.
• Highly phosphorylated forms does not bind to DNA.

IHC Experiment Tips

Precautions

• Ki67 is primarily expressed in proliferating cells and is almost absent in certain normal tissues (e.g., liver). Therefore, it is easier to detect Ki67 in tumor tissues with strong proliferative capacity. Try using H&E staining to distinguish tumor from adjacent tissues to ensure samples contain highly proliferative tissue, thus selecting the right sample can improve experimental results.
• While Ki67 can be detected in proliferating cells, some tissues have low proliferative cell content (e.g., colonic crypt base), resulting in low positive cell proportions or no signal observed in the final section. Consider trying different sections to find a suitable observation field.
• If unsure about the expression of the target protein in your sample, it is strongly recommended to use a positive control in the experiment, such as the germinal center of tonsil tissue.

Positive Controls:

• Human tonsil tissue.
• Rat and mouse spleen tissue.

Example Results

Figure 3: IHC - Anti-Ki67 Antibody (ab16667)
Immunohistochemistry image of Ki67 stained with ab16667 on formalin-fixed, paraffin-embedded mouse spleen tissue sections. Antigen retrieval was performed using sodium citrate buffer (pH 6.0).

Key control points

In the experiment, in addition to paying attention to routine issues, special attention should be paid to the following key control points:

Sample fixation:

1. The optimal fixation time for samples depends on the size and type of tissue, but for most samples, it is advisable to fix with 4% PFA at room temperature for 18-24 hours.
2. Insufficient fixation can result in higher signal at the edges of the sample and weaker or no signal in the center.
3. Over-fixation can mask the epitope. While antigen retrieval can help overcome this masking, if the tissue is fixed for an extended period (e.g. over a weekend), there may still be no signal even after antigen retrieval.

Antigen retrieval:

1. When performing immunohistochemistry experiments on paraffin-embedded sections, antigen retrieval conditions should be optimized. It is recommended to try heat-induced antigen retrieval at 110°C for 15 minutes using a pressure cooker.
2. When performing immunohistochemistry experiments on frozen sections, if the sample is fixed for a long time (such as 18-24 hours) using 4% PFA, it is recommended to use enzyme antigen retrieval or microwave retrieval methods.

Blocking:

1. If using HRP conjugate for detection, treat the sections with 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase activity.
2. If using fluorescent detection by coupled secondary antibodies, it is recommended to add 0.3M glycine to the blocking solution to quench autofluorescence.

ICC Experimental Tips

Precautions

• Ki67 is localized on chromosomes and in the cell nucleus, so it is recommended to fix cells with 4% PFA. Improper fixatives may result in no staining.
• The cellular state can affect proper protein localization; therefore, ensure cells are in a normal state before fixation to avoid mislocalization of the target protein.

Positive Controls

• HeLa cells.
• HAP1 cells.

Example Results

Figure 4: ICC - Anti-Ki67 Antibody (ab16667). Green: Ki67, Red: alpha Tubulin, Blue: DAPI.

Sample name: HeLa cells.
A: HeLa cells fixed with 4% PFA for 10 minutes, followed by permeabilization with 0.1% Triton X-100 for 5 minutes.
B: HeLa cells fixed with 4% PFA for 10 minutes.

Key control points

In the experiment, in addition to paying attention to routine issues, it is also important to focus on the following key control points:

Sample preparation:

1. It is necessary to ensure cells are healthy and growing in a normal state. A confluency of 60%-80% is suitable for most samples.

Sample fixation:

1. Fix Ki67 with 4% PFA, and it is recommended to fix at room temperature for 10-20 minutes.

Sample permeabilization:

1. It is recommended to permeabilize with 0.1-0.25% Triton X-100 at room temperature for 10 minutes.

References

• Li LT, Jiang G, Chen Q & Zheng JN. Ki67 is a promising molecular target in the diagnosis of cancer. Mol. Med. Rep. (2015).11, 1566–1572. doi: 10.3892/mmr.2014.2914.
• Yang C, Zhang J, Ding M, Xu K, Li L, Mao L, Zheng J. Ki67 targeted strategies for cancer therapy. Clin Transl Oncol. (2018). 20(5): 570-575. doi: 10.1007/s12094-017-1774-3.