The Differences Between Immunocytochemistry, Immunohistochemistry, and Immunofluorescence

Immunocytochemistry (ICC), immunohistochemistry (IHC), and immunofluorescence (IF) are techniques that use antibodies to detect cell-associated antigens and provide semi-quantitative data about target protein expression, distribution, and localization. These terms are often confusing and are sometimes mistakenly used interchangeably. Thus, it is important to understand the fundamental differences between these various techniques.

On this Page:

• What Samples Are Used?
• What Labeling Methods Are Best?
• Image Examples
• Additional Resources

Sample Type 

• Immunohistochemistry refers to tissue immunostaining, of either formalin-fixed paraffin-embedded (FFPE) or frozen tissue. 
• Immunocytochemistry refers to the immunostaining of cultured cell lines or primary cells including smears, swabs, and aspirates. 

Sample Preparation

For IHC staining, samples are either embedded in paraffin or frozen to preserve tissue morphology. In addition, both IHC and ICC protocols require sample fixation. Some fixatives, including formaldehyde, crosslink proteins and can mask epitopes and reduce antigen-antibody binding. An antigen retrieval method such as heat-induced (HIER) or proteolytic-induced (PIER) is typically included before IHC staining to restore tissue antigenicity. IHC often requires blocking non-specific binding epitopes during sample preparation.

Labeling Method – IHC and ICC have traditionally used chromogenic reagents to detect target antigens. With this method, an enzyme such as horseradish peroxidase (HRP) is conjugated to the epitope-specific antibody which localizes it to the target site. HRP converts a soluble substrate such as DAB or AEC into a colored precipitate at the antigen site. With IF detection, the detection antibody is conjugated to a fluorochrome which emits light at a specific wavelength following appropriate excitation. Read more about chromogenic vs fluorescent detection.

For both chromogenic and fluorescent detection, the label may be conjugated directly to a primary antibody that binds to the antigen. Frequently, IHC, ICC, and IF use the indirect method of detection in which a secondary antibody, directed against the primary antibody, carries the label. The indirect method is more sensitive than using a directly labeled primary antibody because multiple labeled secondary antibodies can bind to a single primary antibody. See our Secondary Antibody Handbook for more information.

With the extensive list of available fluorescent labels and their ease of use in multiplex applications, researchers are choosing ICC/IF and fluorescence IHC more regularly. Additionally, the use of fluorescent conjugated antibodies specific against organelle markers, facilitates the analysis of proteins of interest and extends multiplex capabilities in both IHC and ICC.