Immunocytochemistry (ICC) Protocol and FAQs

Immunocytochemistry (ICC) is a technique used to study specific proteins, antigens or other biomolecules in cell samples. ICC technology combines the specificity and color reaction of antibodies, which is used to locate and detect the expression of intracellular or cell surface molecules. By using specific antibodies to bind to markers (such as fluorescence or enzyme), ICC can provide high-resolution cell localization information under the microscope, which is widely used in basic research, disease diagnosis and drug.

The Principle of Immunocytochemistry

The basic principle of ICC is the specific binding of antibodies to their target antigens (usually intracellular or cell surface specific proteins). Visualize the distribution and expression of proteins by fluorescence, enzyme, or chemical labeling. Each antibody is a molecule that targets a specific antigen and binds to a specific structural region of the antigen (usually an antigen epitope). When the antibody binds to the target protein, the antigen-antibody complex is formed. Then the antibody-antigen complex was detected by coloration or fluorescence labeling. The detection of these complexes can be observed by a microscope and localized in specific areas of the cell. Commonly used markers include :

Enzyme labeling (such as horseradish peroxidase HRP) : Color reaction occurs after reaction with the substrate.

Fluorescence labeling (such as FITC, Cy3) : After excitation, fluorescence is emitted to facilitate observation under a fluorescence microscope.

The Steps of Immunocytochemistry

Sample Preparation-Cell Culture

Cells were seeded on suitable petri dishes or slides to ensure cell attachment or suspension growth. Cells were fixed with appropriate fixatives (such as 4 % paraformaldehyde or methanol) to maintain cell morphology and the location of target molecules. The fixation time and concentration should be adjusted according to the cell type and the characteristics of the target protein. After that, cells need to be permeabilized. Permeation is to enable the antibody to penetrate the cell membrane to reach the inside of the cell. Commonly used permeabilizers include 0.1 % -0.5 % Triton X-100 or 0.1 % Tween-20. The permeation time is generally 10-15 minutes, adjusted as needed. The non-specific antibody binding sites were blocked with blocking solution (such as 5 % normal goat serum or bovine serum albumin BSA). Closure time is usually 30 minutes to 1 hour, at room temperature.

Antibody Incubation

The primary antibody (specific antibody against the target protein) was diluted in the blocking solution and added to the sample. The incubation time is usually 1 hour to overnight, and the temperature can be adjusted according to the experimental requirements (usually room temperature or 4℃ overnight). If secondary antibodies are used, appropriate secondary antibodies (such as enzyme-labeled or fluorescent-labeled antibodies) need to be selected according to the experimental purpose.

Washing

The samples were washed with PBS or TBS buffer to remove unbound antibodies. Washing steps are usually repeated 3 times, 5-10 minutes each time.

Secondary Antibody Incubation

If the primary antibody-secondary antibody system is used, then the secondary antibody containing the marker is added to the sample. The secondary antibody binds to the primary antibody and provides detectable signals (such as fluorescence or enzyme). The incubation time was 1 hour at room temperature.

Finally Washing

Wash the cells again with PBS or TBS buffer to remove excess secondary antibodies.

Color/Fluorescence Detection

If the enzyme-labeled secondary antibody (such as HRP) is used, the substrate solution can be added, and the enzyme reacts with the substrate to produce a visible color reaction.

If the fluorescently labeled secondary antibody is used, the labeled signal can be directly observed under a fluorescence microscope.

Sealing

Use a sealing solution containing an anti-fading agent to seal the film, prevent the attenuation of the fluorescence signal, and protect the sample for microscopic observation.

Microscopic Observation

The cells were observed by optical microscope or fluorescence microscope to analyze the localization and distribution of the target protein in the cells.

Note 1 Antibody selection : Choosing the appropriate and specific antibody is the key to the success of ICC experiment. Ensure the effectiveness and specificity of the antibody in the target cells.
Note 2 Fixation method : Different fixatives have different effects on protein and cell structure. Choosing the appropriate fixative is crucial for retaining the antigen epitope.
Note 3 Selection of fluorescence labeling / enzyme labeling : fluorescence or enzyme labeling was selected according to the needs of the experiment. Fluorescence labeling was suitable for multiple labeling experiments, and enzyme labeling was suitable for chromogenic detection.

Figure 1. Absorption control is the incubation of the primary antibody with the antigen used to generate the antibody. (A) The primary antibody incubated with excess antigens binds all of the Fab sites capable of binding the antigen in the tissue (arrow). (B) If the correct antigen and an incorrect antigen have the same epitope (arrows), then binding to both is inhibited by the absorption control. (C) In some cases, the antigen adsorbed by the antibody binds to proteins in the tissue, and the adsorbed antibody appears to bind to a protein. (Reference source: Controls for Immunocytochemistry.)

Advantages of Immunocytochemistry

High specificity : it can detect specific proteins, especially for localization and quantification of proteins.

Cell-level resolution : Subcellular localization of intracellular proteins can be observed.

Quantitative and qualitative analysis : Combined with image analysis software, the protein expression level can be quantified and the changes of protein distribution under different conditions can be analyzed.

Application of Immunocytochemistry

Protein Localization

Localization of a specific protein in a cell (e.g., cell membrane, cytoplasm or nucleus).

Drug Screening

ICC can be used to observe the effect of drugs on specific molecules or structures of cells, providing support for drug development.

Disease Research

Immunocytochemistry is often used to observe the expression and distribution of disease-related proteins in cancer, neurodegenerative diseases and other studies.

Alpha Lifetech provides Immunocytochemistry (ICC) services, adheres to customer demand-oriented, relies on advanced technology platforms, and provides comprehensive and customized scientific services to ensure the smooth progress and high-quality completion of the project.

FAQ

1. Weak signal or no signal.

Cause : (1) Poor antibody quality : The antibody used may not be very specific or ineffective. (2) Inappropriate antibody concentration : too low or too high antibody concentration may lead to weak signal or background noise. (3) Fixation process issues : If the cell is not fixed well, the target protein may lose its detectability. (4) Poor penetration : The cell membrane may not be permeable enough, resulting in antibodies unable to enter the cell. Solutions : (1) Use high-quality, validated antibodies. (2) The concentration was adjusted according to the antibody instructions, and the concentration gradient experiment was carried out. (3) Select the appropriate fixing agent (such as 4 % formaldehyde) and ensure that the fixing time and temperature are appropriate. (4) If you need to label the protein inside the cell, use appropriate penetrants (such as 0.1 % Triton X-100) to increase the permeability of the cell membrane.
2. High background signal.

Cause: (1) Non-specific binding : The antibody may bind to other non-target molecules in the cell. (2) Secondary antibody problem : the quality of secondary antibody is not good or not suitable for cell type. (3) Inadequate washing : The antibody is not completely washed away, resulting in increased background signal. Solutions : (1) Increase the blocking step by using the appropriate blocking solution (such as 5 % bovine serum albumin or normal goat serum). Select the appropriate secondary antibody and ensure that it matches the species of the primary antibody. (2) More washing buffers are used in the washing step to ensure that the antibody and non-specific binding substances are fully removed.
3. Loss or damage of cell morphology.

Cause: (1) Improper fixation steps : Excessive or insufficient fixation can lead to changes or loss of cell morphology. (2) Improper use of penetrant : Too much or too strong penetrant will destroy the cell structure. Solution : (1) Ensure that the fixation steps are performed according to the standard, usually using 4 % formaldehyde or other suitable fixatives. (2) Avoid too many penetrants, adjust the concentration of penetrants or use different kinds of penetrants.
4. Uneven dyeing.

Cause: (1) Uneven cell distribution : sample treatment is not uniform, cell distribution may be uneven. (2) Improper dyeing time : dyeing time is too short or too long, resulting in uneven dyeing. Solutions : (1) Ensure that the cells are evenly coated on the slide. (2) The staining time and antibody incubation time were optimized to ensure uniform reaction.
5. Specificity of antibodies.

Cause: Antibodies may cross-react with non-target proteins in other cells. Solutions : The verified antibodies were used and appropriate positive and negative controls were performed. Try using other antibodies or pre-adsorbing antibodies to reduce non-specific binding.

reference

[1] Maxwell P, Salto-Tellez M. Validation of immunocytochemistry as a morphomolecular technique. Cancer Cytopathol. 2016;124(8):540-545. doi:10.1002/cncy.21692

[2] Kanber Y, Pusztaszeri M, Auger M. Immunocytochemistry for diagnostic cytopathology-A practical guide. Cytopathology. 2021;32(5):562-587. doi:10.1111/cyt.12993

[3] Jain D, Bubendorf L. Immunocytochemistry in cytology: myth or reality. Acta Cytol. Published online January 30, 2025. doi:10.1159/000543867

[4] Happonen RP, Heikinheimo K. Introduction to immunocytochemistry. Proc Finn Dent Soc. 1989;85(2):61-67.

[5] Skoog L, Tani E. Immunocytochemistry: an indispensable technique in routine cytology. Cytopathology. 2011;22(4):215-229. doi:10.1111/j.1365-2303.2011.00887.x

[6] Burry RW. Controls for immunocytochemistry: an update. J Histochem Cytochem. 2011;59(1):6-12. doi:10.1369/jhc.2010.956920