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Overview of Immunocytochemistry

Jump to Antibodies


Immunocytochemistry (ICC) is a technique used to label a specific biomolecule within a cell that can be observed through microscopy. The prefix “immuno” serves to indicate that the labeling method involves immunostaining and the use of high affinity antibodies to label proteins or other biomolecules. The visual identification of the protein allows the researcher to confirm the presence of the protein and determine subcellular localization of the protein.


What is Immunocytochemistry?


The similarity of ICC to immunohistochemistry (IHC) often leads to confusion over which term to use. Both methods allow visualization of specific cellular proteins by various schemes of antibody detection. The difference between the two methods is in type of sample used. IHC uses tissue isolated from an organism that is fixed, sliced into thin sections and placed onto slides. By contrast, ICC uses cells that are physically or enzymatically separated from their native tissue; cells that are normally suspended in body fluids such as blood, or cell lines grown in a laboratory. Cells that originate from body fluids or grown in suspension need to be placed on slides by methods like smears or by centrifuging the cells onto the slides. Adherent culture cells can be grown directly on slides, optically clear plastic dishes or coverslips. Regardless of the source of the cells or how they are grown, at the end of processing for IHC and ICC the sample undergoes immunostaining and microscopic observation.


How Does It Work?


ICC protocols can be divided into three major segments: sample processing, immunostaining/detection and observation. The processing steps prepare the cells to be immunostained. It is common to use fixative reagents for ICC such as paraformaldehyde (PFA), which helps cells retain their morphology and adhere to the slides during subsequent steps. The immunostaining steps begins by incubating the sample with a primary antibody that detects a specific protein target. There are several methods of detecting the binding of the primary antibody to the target protein. The original method of detection (the reason for chemistry in the ICC) uses a second antibody, which is conjugated to an enzyme such as horseradish peroxidase and recognizes the primary antibody. In this case, horseradish peroxidase deposits a colored reaction product in areas where the primary antibody has bound.

There are many variations for the immunostaining and detection methods. The process described above is an indirect detection method because an additional antibody is used to visualize the protein (the HRP enzyme is on the secondary antibody). If the enzyme is conjugated to the primary antibody with no need for additional antibodies, the term direct detection applies. The ability of synthesize fluorescent compounds that can be conjugated to antibodies gave rise to another detection system, termed as immunofluorescence (IF). The IF detection method is extremely sensitive and allows multiple proteins to be visualized at once.


Why Immunocytochemistry?


ICC is a powerful technique for visualizing proteins and biomolecules within cells. The procedure is amenable to many assays including confirming the presence/absence of a tumor biomarker or studying complex protein interactions. The ability to perform multiplex immunostaining facilitates rapid detection of signaling pathway components or to elucidate transcription networks. Due to the variety of ICC assays and detection methods, antibody performance in ICC is critical.


Recent Immunocytochemistry Citations Using Bethyl Antibodies


Bethyl Laboratories sells high quality ICC antibodies that are validated on-site by our own scientists. These products have recently been used to study:

  • Hundreds of RNA-binding proteins associated with cancer, neurobiology, and development1
  • The effects of failed DNA replication on mutation rates and cancer predisposition2
  • Proteins typically responsible for spindle formation creating an environment for cancerous cell division3
  • The complex signaling network responsible for resolving double-stranded breaks in DNA4
  • The role of a previously understood nuclear protein associated with a number of brain-related pathologies5



Thousands of Bethyl antibodies are validated for use in immunochemistry. The complete list is here:




1. Sundararaman B, Zhan L, Blue SM, Stanton R, Elkins K, Olson S, Wei X, Van Nostrand EL, Pratt GA, Huelga SC, et al. 2016. Resources for the comprehensive discovery of functional RNA elements. Mol Cell. Mar 17; 61(6): 903-913. [330 Bethyl antibodies were used; please refer to the article for the complete list]

2. Renaud E, Barascu A, Rosselli F. 2016. Impaired TIP60-mediated H4K16 acetylation accounts for the aberrant chromatin accumulation of 53BP1 and RAP80 in Fanconi anemia pathway-deficient cells. Nucleic Acids Res. Jan 29; 44(2): 648–656. [Bethyl antibody used: RIF1 Antibody (A300-569A). Please note that this antibody has not been validated by Bethyl for use in ICC and as such Bethyl cannot guarantee results as published in this paper.]

3. Chavali PL, Chandrasekaran G, Barr AR, Tátrai P, Taylor C, Papachristou EK, Woods CG, Chavali S, Gergely F. 2015. A CEP215-HSET complex links centrosomes with spindle poles and drives centrosome clustering in cancer. Nat Commun. Mar 18;7:11005. [Bethyl antibody used: KIFC1 Antibody (A300-952A). Please note that this antibody has not been validated by Bethyl for use in ICC and as such Bethyl cannot guarantee results as published in this paper.]

4. Hartlerode AJ, Morgan MJ, Wu Y, Buis J, Ferguson DO. 2015. Recruitment and activation of the ATM kinase in the absence of DNA-damage sensors. Nat Struct Mol Biol. Sep;22(9):736-743. [Bethyl antibody used: Phospho KAP-1 Antibody (A300-767A)]

5. Carmona-Mora P, Widagdo J, Tomasetig F, Canales CP, Cha Y, Lee W, Alshawaf A, Dottori M, Whan RM, Hardeman EC, et al. 2015. The nuclear localization pattern and interaction partners of GTF2IRD1 demonstrate a role in chromatin regulation. Hum Genet. Oct;134(10):1099-1015. [Bethyl antibody used: ZNF198 Antibody (A301-710A). Please note that this antibody has not been validated by Bethyl for use in ICC and as such Bethyl cannot guarantee results as published in this paper.]