Immunofluorescence, Immunocytochemistry and Immunohistochemistry – what’s the difference?

The terms immunofluorescence, immunocytochemistry and immunohistochemistry are often used interchangeably. They are however clearly distinct from one another, and it is important that they are used appropriately to avoid confusion. The prefix, “immuno”, refers specifically to the binding of an antibody to an antigen, while the suffix confers different meanings:

  • Immunofluorescence (IF) is a detection method, during which antibody binding to an antigen is visualized using a fluorophore
  • Immunocytochemistry (ICC) is a technique; the term refers to the antibody-based detection of an antigen in cells (“cyto”)
  • Immunohistochemistry (IHC) is a technique; the term describes the antibody-based detection of an antigen in tissues (“histo”)

Immunofluorescence has traditionally relied on the use of secondary antibodies which have been labeled with fluorescent dyes or fluorescent proteins, however labeled secondary antibodies can be a common source of unwanted background signal. To circumvent this issue, many researchers prefer to instead use directly labeled primary antibodies for detection. These offer several clear advantages:

✓ Non-specific binding is avoided since secondary antibodies are not used
✓ Multiplexing is possible with antibodies from the same species
✓ Faster since there is no secondary antibody incubation step and therefore fewer wash steps
✓ Data quality is improved through assay simplification

Unfortunately, many commercial antibody suppliers often do not offer the required antibody labeled with the fluorophore of choice. Furthermore, direct labeling of primary antibodies with fluorophores can be a complicated and time-consuming process which requires specialist knowledge. To overcome these issues we offer Lightning-Link®, an innovative technology that enables direct labeling of antibodies, proteins, peptides or any other biomolecule which has free amine groups, with just 30 seconds hand-on time.

Lightning-Link process diagram

The Lightning-Link® conjugation process. The antibody is added to the vial of freeze-dried Lightning-Link® label, incubated, and the conjugate is then ready to use. With no separation steps, antibody recovery is 100%.

To find out more about Lightning-Link®, why not watch our video?

The Lightning-Link® product range includes kits for labeling antibodies with a wide range of fluorophores, covering the spectrum from UV to far infra-red, and is ideally suited for applications which rely on an immunofluorescent readout. When performing a multiplexing experiment, it is important to choose dyes that do not overlap spectrally to avoid background signal, and the extinction coefficient and Stokes shift data should also be taken in to account. We’ve made label selection simple by summarizing these parameters for all our fluorescent labels in an easy to use table.

Download our fluorescence table here!

Immunocytochemistry provides a clear visual representation of cellular protein localization although, as with any experiment, the inclusion of relevant controls is critical to correct interpretation of the data. Western blotting is a reliable and commonly-used method of demonstrating the specificity of the primary antibody. Another useful control is to stain the sample with two antibodies against different epitopes of the same protein to check for colocalization; direct antibody labeling is particularly beneficial to this approach, especially if both primary antibodies have been raised in the same host species.

Immunocytochemistry - Lightning-Link staining

ICC data generated using Lightning-Link®. RAW264 macrophage cell line stained withanti-F4/80 antibody conjugated to Fluorescein using Lightning-Link®, counter-stained with DAPI.

In addition to our fluorescent labeling kits, the Lightning-Link® product range also includes kits for labeling antibodies with Horseradish peroxidase (HRP), Alkaline Phosphatase and Glucose Oxidase. These enzymes are routinely used for IHC, during which an appropriate substrate is added to the samples to allow the production of a colorimetric readout.

Immunohistochemistry - Lightning-Link staining

IHC data generated using Lightning-Link®. Paraffin-embedded human bone marrow stained with anti-human CD20 (clone #L26) antibody conjugated to HRP using Lightning-Link®.

If you have any questions about our Lightning-Link® technology and how you can use it to streamline your own immunofluorescence, immunocytochemistry or immunohistochemistry experiment, please contact us.