Planning an immunocytochemistry (ICC) project can be tricky as it requires careful selection of compatible antibodies, fluorochromes, and blocking reagents. In this article our scientists have provided a step-by-step guide for single staining of iPSCs, plus trouble-shooting tips to optimize your results.
Planning your immunocytochemistry experiment
Step 1: Decide what pluripotency markers you want to visualize
Before beginning this project, you will need to determine which pluripotency markers you want to visualize, and whether these are located on the surface or inside of the cell. We have drafted some of the most popular pluripotency markers and their cellular locations in the table below:
Intracellular or surface marker?
REPROCELL Catalogue No.
Step 2: Plan your experiment
If you are planning on staining two markers simultaneously, you should ensure one of your chosen markers is intracellular, and the other is extracellular. This is because it can be difficult to distinguish two markers using ICC if they share the same cellular location.
You should also ensure that the primary antibodies you select are from different species and that the secondary antibodies you choose are conjugated to different colored fluorophores.
As this can be a quite complex stage of planning, it is helpful to use an excel sheet to match antibodies and markers. We have created one in our ICC Starter Pack which includes a tried-and-tested example of antibodies you can stain together. In this pack, we have also included an experimental checklist for you to complete, to ensure you have covered all the necessary steps before beginning your experiment.
Figure 1: Image of iPSCs stained for pluripotency markers Nanog (intracellular, green) and TRA-1-60 (extracellular, red) in REPROCELL's Glasgow laboratory. Image taken at 10X magnification.
Step 3: Order reagents
Once you have determined what antibodies you are going to use, you can begin ordering experimental reagents. Make sure to risk assess your project and complete the necessary COSHH forms before beginning any laboratory work. You could use a tool like this one provided by the HSE to get started.
Also make sure that you have all the necessary laboratory equipment, and that you include negative and positive controls in your analysis.
Figure 2: Image of iPSCs stained for pluripotency markers OCT4 (intracellular, green) and SSEA4 (extracellular, red) in REPROCELL's Glasgow laboratory. Image taken at 10X magnification.
Characterizing your stem cells using ICC
Now that you are ready to execute your experiment, follow our 15-step immunocytochemistry protocol to achieve the results you need. In our ICC Starter Pack, we have included a single and double staining procedure, dependent on your experimental needs.
Below, we have detailed the methodology included in our double staining ICC protocol.
Immunocytochemistry protocol for double staining
- Seed and culture cells in a 24-well plate until ready for ICC analysis. We recommend 30-40% confluency for iPSCs.
- Wash each well 3 times with 0.5 ml of room temperature PBS.
- Fix each well by adding 0.25 ml of 4% paraformaldehyde in PBS and incubating for 20 minutes at room temperature.
- Aspirate the 4% paraformaldehyde and then wash each well 3 times with 0.5 ml of PBS for 5 minutes with gentle agitation. Fixed cells may be stored in 1mL of PBS at 4°C overnight. Note: If visualizing intracellular markers perform this step. Otherwise proceed to step 7.
- Aspirate the PBS and add 150 μL 0.2% Triton-X-PBS to each well. Incubate for 10 minutes at room temperature.
- Aspirate the 0.2% Triton-X-PBS and wash the wells with 0.25 mL PBS per well.
- Add 0.5 ml blocking buffer per well and incubate at room temperature for 1 hour.
Access the full protocol here →
- Dilute the primary antibodies in blocking buffer according to the manufacturer’s instructions.
- Aspirate the blocking buffer and add 250 µl of each diluted primary antibody to each well. Incubate at 4°C for at least one hour. We recommend incubating overnight.
- After the incubation time, wash each well 3 times with 0.5 ml of PBS for 10 minutes with gentle agitation.
Note: If using a conjugated antibody, skip steps 11 through 14 and go directly to step 15. If using a purified primary antibody, continue to step 11.
- Dilute the fluorophore-conjugated secondary antibodies in blocking buffer, according to the manufacturer’s instructions, and add 250 µL of each antibody to each well.
- Incubate at room temperature for one hour, protecting the plate from light.
- Following incubation, wash each well 3 times with 0.5 ml of PBS for 10 minutes with gentle agitation.
- Prepare a 2 µg/ml working solution of DAPI by diluting in PBS. Add to each well and incubate for 10 minutes at room temperature.
- Wash each well once with 0.5 ml of PBS for 5 minutes with gentle agitation.
- Aspirate any remaining PBS and add 1 to 2 drops of mounting medium to each well to stain the nuclei and preserve the samples for fluorescence microscopy imaging. Alternatively prepare a 0.2 µg/mL DAPI solution to stain the nuclei and visualize the cells.
- Analyze cells using a microscope with image capturing abilities and annotate the images as follows:
Figure 3: Characterization of fibroblast and blood derived iPSCs using single staining. These iPSCs were characterized by expression of intracellular pluripotency markers NANOG (A) and OCT4 (B). First row (A): Fibroblast derived iPSCs; Second row (B): Blood derived iPSCs. First column: Phase contrast image of iPSCs; Second column: Visualization of cell nuclei (blue: DAPI); Third column: visualization of intracellular markers (green: DyLight 488); Fourth column: merge of the second and third columns. All images were taken at 10x magnification.
Figure 4: Characterization of fibroblast derived iPSCs using single staining. These iPSCs were characterized by expression of cell surface pluripotency markers TRA-1-60 (A) and SSEA-4 (B). First column: Phase contrast image of iPSCs; Second column: Visualization of cell nuclei (blue: DAPI); Third column: Visualization of cell surface markers (red: Alexa Fluor 594); Fourth column: Merge of the second and third panels. All images were taken at 10x magnification.
ICC Troubleshooting Tips
High background signal
If you are experiencing a high background signal, this means that your antibodies are binding to non-specific sites. There are a few steps you can take to reduce a high background signal.
- Increase the number of washes you perform between incubation. You can also increase the length of these washes e.g. primary or secondary antibody.
- Increase the percentage of serum in your blocking buffer e.g. if you were using 10% animal free blocker, increase this to 15% or more.
- Reduce the concentration of antibody used during incubation e.g. if you were using 0.1% antibody diluted in blocking buffer, try 0.05%.
- If all else fails, try switching from a polyclonal antibody to a monoclonal antibody to reduce cross-reactivity. REPROCELL sell a number of monoclonal antibodies, which you can view in our catalog.
Figure 5: An example of an ICC image with high background signal. High background can be reduced by increasing the concentration of blocking buffer, increasing antibody concentration or switching to monoclonal antibodies.
Weak fluorescence signal
A weak fluorescence signal can usually be amended by taking the exact opposite steps to the above.
- Reduce the number of washes you perform between incubation or decrease the length of these washes.
- Reduce the percentage of serum in your blocking buffer, or switch to an animal free blocking agent.
- Increase the concentration of antibody used during incubation e.g. if you were using 0.1% antibody diluted in blocking buffer, try increasing this to 0.2%.
- If you are staining for an intracellular marker, ensure that you have permeabilized the cell membrane using Triton X-100 or a similar reagent.
Figure 6: An example of an ICC image with reduced fluorescence signal. The signal can be increased by increasing the antibody concentration, increasing the blocking buffer concentration or switching to an animal free blocking buffer.
Let us know how you get on
Taking on an ICC project can be daunting – especially when multiple antibodies and markers are involved. Hopefully, the information and resources we have provided will leave you feeling more confident about executing your ICC experiment. Let us know how you get on in the comments below, and follow us on LinkedIn for more stem cell articles.
REPROCELL stem cell characterization services
As part of our contract services at REPROCELL, we offer induced pluripotent stem cell (iPSC) characterization by ICC – meaning you don’t need to spend valuable time and resources planning and optimizing your characterization project.
Visualize several pluripotency markersWe can visualize a range of intracellular and cell surface pluripotency markers including the intracellular markers OCT4, NANOG and SOX2, plus surface markers TRA-1-60, TRA-1-81, SSEA-3 and SSEA-4. Stem cell nuclei are counterstained with DAPI, and a green Stemgent® secondary antibody is used to visualize the pluripotency markers.
Perform double and single stainingWe also provide a double staining service where intracellular and cell surface markers are visualized simultaneously. This means that you can track the expression of two separate pluripotency markers in the same stem cell line at the same time.
Work with a dedicated study directorWe will assign your project a dedicated study director who will stay in contact throughout the duration of your custom project. If you have any questions or concerns about your study, they will be happy to answer any questions that you have about the work being carried out for you.
- History of Immunohistochemistry in Pathobiology of Human Diseases: A Dynamic Encyclopedia of Disease Mechanisms (2014)
Note: This blog post was originally published in March 2018 but has been updated for accuracy and clarity.