I want to use Ki67 as a proliferation marker. I tested the antibody with small intestine tissue, but I can only detect Ki67-positive cell at lymphoid nodule. No positive cells were detected at mucosa epithelium. This staining results confused us. I am wondering whether I need to treat with Triton X-100. Is there anyone have experience about Ki67 immunostaining?
Here is more detail of my staining condition:
Sample: formalin-fixed, paraffin-embedded specimens
Primary Antibody: DAKO anti-human Ki67 Clone MIB-1
Antigen retrival: Cook in 10mM Citric buffer pH6 for 20 min
Dilution&Incubatio: 1:50 4℃ overnight in humid chamber
I have experience with the Ki67 from abcam (ab15580).
I have used this antibody to stain proliferating cells within 4% para fixed and frozen mouse spinal cord mounted on glass slides. The staining procedure did not require any antigen retrieval.
This is a rabbit polyclonal Ab, reactive with Ms Hu and Hrs, used antibody at 1:500 diluted in the blocking buffer. I have attached the product sheet for your information. I would be happy to send an outline of the staining procedure I used if you feel it would be helpful.
You should contact the company and ask for a sample aliquot of the antibody to test before purchase.
In my lab MIB-1 is successfully used with the antigen retrival buffer EDTA pH8.0, we perform it using microwave oven (usually 3x5' cicles, in case where reactive is weak we perform 2x5' al max Watt and 3x5' al 750 Watt)
I agree with Ahed. Citrate buffer should work fine, but do it in a pressure cooker (decloaking chamber) to get better results. Our lab uses 10 minutes, but I could see it working in less time. Try a range of dilutions with both citrate buffer and the 8.0 pH EDTA buffer using 10 minutes in the pressure cooker to find the optimum choice.
I have experience with the Ki67 from abcam (ab15580).
I have used this antibody to stain proliferating cells within 4% para fixed and frozen mouse spinal cord mounted on glass slides. The staining procedure did not require any antigen retrieval.
This is a rabbit polyclonal Ab, reactive with Ms Hu and Hrs, used antibody at 1:500 diluted in the blocking buffer. I have attached the product sheet for your information. I would be happy to send an outline of the staining procedure I used if you feel it would be helpful.
You should contact the company and ask for a sample aliquot of the antibody to test before purchase.
The nuclear antigen Ki-67 is a proliferation marker expressed only in cycling cells. A strong correlation between S-phase fraction and Ki-67 index has been seen . Consequently, quantitative assessment of Ki-67 staining on paraffin-embedded tumour sections provides an accurate estimate of the proliferation index of individual tumours. Cytotoxic chemotherapy induces programmed cell death by apoptosis. The percentage of apoptotic cells in tumour sections may be measured by labeling fragmented DNA breaks and calculating the apoptotic index (AI) using the TUNEL (terminal transferase uridyl nick-end labeling) assay .
antigen retrieval technique for Dako, i did it will microwave, and it worked perfectly fine in two cycles each of i guess 3x5 at 750 watts, but i used cytokeratin AE1/AE3, S-100, ER/PR and p 63, not Ki-67, For many antibodies you may not requie antigen retrieval technique, so it can be a case with Ki-67. UltraVision LP detection system is one of the few new companies and they provide very good diagnostic kit which does not require antigen retrieval step, i have used it and is very good product of Thermo scientific company.
Primarily, a vendor has previously optimized their antibodies with their own detection system; typically they test this way as part of their quality plan. That is not to say that they can’t be optimized with an alternate vendor systems. The LP detection (TL-015-AF) from LabVision is a dual polymer recognizing both mouse and rabbit and can be used RT for approximately 30-60 minutes incubations. So, most Rabbit and mouse host antibodies will be detected with this detection assuming the antibody concentration and incubation is optimized.
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