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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Ki67 Cell Proliferation Assay
- Ki67 Cell Proliferation Assay (Immunocytochemistry)
- Rinse the cell cultures with 0.1 M phosphate (PO4) buffer for one minute two times. Fix the culture with 4% paraformaldehyde (PFA) for 20 min at room temperature. Remove the PFA and rinse the wells with PBS for seven minutes three times.
- After the final rinse, add 100 µl of blocker solution (phosphate buffer saline, 5% normal donkey serum, 0.4% bovine albumin serum, and 0.2% Triton X-100) to each well and incubate at room temperature for 1 hr. Prepare the primary antibody, rabbit anti-Ki67, by diluting in blocker solution at a working ratio of 1:200.
- Remove the blocker solution and apply 100 µl of the primary antibody solution to each well. Cover the 96-well plate and incubate the samples at 4 °C overnight.
- On the following day, remove the antibody solution and rinse with PBS for 7 min, 3 times.
- Prepare the secondary antibody, donkey anti-rabbit Cy3 in blocking solution at a working ratio of 1:500. Add DAPI nuclear stain to the secondary antibody solution at a dilution of 1:100. After the removal of the last PBS rinse, apply 100 µl of the secondary antibody/DAPI solution to each well. Incubate at room temperature in the dark for 90 min.
- Remove the secondary antibody/DAPI solution and rinse each well with PBS for 7 min, 3 times. Cover the 96-well plate and store at 4 °C until imaging.
2. Automated Imaging and Multiwavelength Scoring Analysis
- Load a 96-well plate (previously processed for Ki67 immunolabeling) into the HCS system and allow the plate to equilibrate for 20 min. Open the HCS system image acquisition and analysis software.
- Choose the acquisition settings for the 10X objective using camera binning at 1 and a gain setting of 2. Find the Z-plane in which the cells reside by utilizing the Auto Exposure function and calculate the offset for each wavelength of interest. For this analysis, capture images for DAPI (W1), Cy3 (W2). Choose the maximum intensity level at which the negative control wells show no signal for image acquisition. Confirm this setting is appropriate for the positive wells.
- Acquire plate. Capture images and store them in the image acquisition and analysis software’s database.
- Once images have been acquired, open image acquisition and analysis offline software and review plate data from the images acquired above.
- Select the Multi-wavelength scoring analysis. Configure the minimum and maximum intensities for each wavelength.
NOTE: DAPI detection should mark the staining around each visible nucleus. Cy3 should detect the positive cells with Ki67 immunoreactivity (IR) and should not detect IR under the negative controls. For Cy3 Ki67 IR, the approximate minimum width was 7 µm, approximate maximum width was 30 µm, the intensity above the local background was 150 gray levels, and the minimum stained area was 50 µm2. - Run analysis for all positions. Export the data to view in spreadsheet.