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Immunocytochemistry - Protocol & Troubleshooting

Immunocytochemistry (ICC) is a technique for assessing the presence of specific proteins or antigens in cells through the use of labeled antibodies. It is the product of combining immunofluorescence techniques with morphological techniques. We describe the optimized protocol for ICC and the procedure for the detection of antigens by antibodies in the cellular environment.

Creative Biolabs provides our customers with specific ICC protocols and troubleshooting, which is applied to study the presence of proteins and subcellular localization.

Solutions and Reagents

Stages Solutions and Reagents
Sample Preparation Phosphate buffer (PBS), paraformaldehyde, permeabilization buffer
Antibody Labeling Blocking buffer, primary antibody, antibody dilution buffer, PBS, secondary antibody
Dyeing Streptavidin peroxidase conjugated, hematoxylin/acetic acid, ultra-pure sterile water, coverslip solution

Immunocytochemistry Procedure


1. Cell Culture and Fixation

Seed cells on sterile glass coverslips or tissue culture plates, and add fresh medium. Allow cells to grow to the desired density before labeling. Then fix cells with appropriate fixation methods, wash them, and set them aside.

2. Permeabilization and Blocking

Incubate the slides with a permeation medium for 10-15 minutes to permeate the membrane. Wash slide when finished to remove the permeate. Then Block the sample using the blocking buffer and wash it for use.

3. Antibody Labeling

Dilute the primary antibody to the recommended concentration with blocking buffers. Then add the primary antibody to the slide and incubate at room temperature, wash in PBS when finished.

Prepare appropriately diluted fluorescent dye-coupled secondary antibodies. Then add secondary antibodies to slide and incubate at room temperature in a dark environment. And wash gently in PBS upon completion.

4. Repainting and Observation

Blocking can prevent non-specific binding of the antibody to the membrane. Place the membrane in the blotting vessel, add enough blocking solution to cover the entire membrane surface, and shake to incubate. This can help to reduce background noise while enhancing the target protein signal.


You can browse our ICC troubleshooting tips and advice below, which focuses on the causes and possible solutions to common problems in ICC assays.

No staining or faint staining

  1. Cell causes. First, target proteins may not be present or may be present at very low levels in the cell sample. Second, cells may also be detached from the coverslip or over-fixed. Cells can be made to adhere by reducing the intensity of the wash, using another fixation solution, or lengthening/reducing the fixation time. It is also possible that the cells are not fully permeabilized and you can use other permeabilizing solutions to improve. Cell drying will not show a fluorescent signal, so it is important to keep the sample in liquid throughout the procedure.
  2. Antibody causes. You may not be using enough primary or secondary antibodies. So, you can adjust the antibody dilution to determine the optimal antibody concentration or increase antibody incubation time. Antibodies may not be compatible. You should ensure that secondary antibodies are produced against the animal that produced the primary antibody in order to achieve synergy. If using fluorescent assays, you should ensure that the coupled fluorescent-stained antibodies are not exposed to light.
  3. Chromogen and substrate causes. Improper use of chromogen and substrate can cause this. You can choose another suitable chromogen and substrate and repeat the test. It is also possible that the re-staining agent is over-stained and the re-staining time can be reduced.
  4. Microscope causes. Check and adjust the microscope settings, increase the exposure time of the camera and observe if the situation can be improved.

Messy staining results

  1. Antibody causes. The antibody concentration is too high so you may switch the antibody to a lower concentration in the test. And primary antibodies should be from a different species than the cells. Please check if you are using recombinant antibody solutions that may form aggregates. We recommend centrifuging all antibody preparations prior to use.
  2. Cell causes. Disrupted cell morphology or the creation of air bubbles can lead to signal clutter. Keep the cells at high humidity to avoid them drying out as much as possible.

High background

  1. Antibody causes. If you are experiencing a high background signal, it is possible that the antibody is binding to a non-specific site. There are steps you can take to reduce the high background signal. You can titrate the antibody to determine the optimal concentration to promote the reaction of primary and secondary antibodies, increase the concentration of the blocking buffer, or switch to a monoclonal antibody to reduce the high background.
  2. Fixation causes. Too long a fixation time can affect epitopes and lead to high background levels. Try using shorter exposure times, lower temperatures, lower concentrations of fixative, or reducing the length of fixation.
  3. Blocking agent causes. Insufficient closure leads to high background. You can increase the closure latency and consider replacing the blocking agent.
  4. Incubation causes. Incubate for too long or at too high an incubation temperature.
  5. Washing causes. It is critical that each step needs to be washed correctly and adequately between steps. When a high background occurs, you can increase the amount of wash, gently agitate the wash, or increase the concentration of Tween in PBS.
  6. Contamination causes. You need to make sure that the slides are clean and free of dust. The buffer should be kept fresh to prevent microbial contamination.

If you are having difficulty with a specific ICC operation, please first review the common problems listed above. If your question is not answered, please fill out the form in as much detail as possible and contact us. Our technical support will contact you shortly to take your comments seriously and help resolve the problem.

ICC is a relatively simple and straightforward experimental approach. For each ICC study, we can help you identify and optimize variables and troubleshoot various factors. For further services and assistance, please contact us.


  1. Dey P. Immunocytochemistry in Histology and Cytology. Basic and Advanced Laboratory Techniques in Histopathology and Cytology. Springer, 2018.
  2. Dey P. Immunocytochemistry in Diagnostic Cytology. Jaypee Brothers Medical Publishers, 2021.

For research use only. Not intended for any clinical use.

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