Planning an Immunofluorescence (immunocytochemistry) project can be tricky. It requires careful selection of compatible antibodies, fluorochromes, and blocking reagents, plus optimization of results if there is a high background signal.
In this article, our scientists describe how immunofluorescence can be used to identify pluripotency markers, and provide troubleshooting tips to optimize your analysis. You can download our immunocytochemistry protocols in our free ICC starter pack below.
Planning your immunocytochemistry analysis
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?
If you are planning on staining two markers simultaneously, you should ensure that one is intracellular, and the other is extracellular. This is because it can be difficult to distinguish two markers using immunofluorescence if they share the same location.
You should also ensure that the primary antibodies you select are from different species (e.g. rabbit and mouse) and that the secondary antibodies you choose are conjugated to differently 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.
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.
Immunocytochemistry protocol for cultured cells
Now that you are ready to execute your experiment, follow our 15-step protocol below to achieve the results you need. In our ICC Starter Pack, we have included both a single and double staining procedure, dependent on your analytical needs. Below, we have detailed the methodology included in our double staining procedure, available for download here.
Stage 1: Adding Primary Antibodies
1. Seed and culture cells in a 24-well plate until ready for ICC analysis. We recommend 30-40% confluency for iPSCs
2. Wash each well 3 times with 0.5 ml of room temperature PBS
3. Fix each well by adding 0.25 ml of 4% paraformaldehyde in PBS and incubating for 20 minutes at room temperature
4. 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.
5. Aspirate the PBS and add 150 μL 0.2% Triton-X-PBS to each well. Incubate for 10 minutes at room temperature
6. Aspirate the 0.2% Triton-X-PBS and wash the wells with 0.25 mL PBS per well
7. Add 0.5 ml blocking buffer per well and incubate at room temperature for 1 hour
8. Dilute the primary antibodies in blocking buffer according to the manufacturer’s instructions.
9. 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.
Stage 2: Adding Secondary Antibodies
10. 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.
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.
12. Incubate at room temperature for one hour, protecting the plate from light.
Stage 3: Visualizing cell nuclei with DAPI
13. Following incubation, wash each well 3 times with 0.5 ml of PBS for 10 minutes with gentle agitation.
14. 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.
15. Wash each well once with 0.5 ml of PBS for 5 minutes with gentle agitation.
16. 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.
How to interpret immunocytochemistry data
Immunocytochemistry troubleshooting tips
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. A weak fluorescence signal can usually be amended by taking the exact opposite steps.
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 3: 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 immunofluorescence signal
- 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 4: 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.|
Stem cell characterization services
As part of our contract services at REPROCELL, we offer induced pluripotent stem cell (iPSC) characterization by immunofluorescence – meaning you don’t need to spend valuable time and resources planning and optimizing your characterization project.
Visualize several pluripotency markers
We 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 staining
We 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 director
We 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.
Figure 5: 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.
How did you get on?
Taking on an immunofluorescence 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 immunofluorescence experiment. Let us know how you get on in the comments below, and follow us on LinkedIn for more stem cell articles.
- 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.