Fluorescence detection is emerging as a popular alternative to traditional chromogen-based IHC. Researchers can generate high-resolution images for protein localization studies and now have the ability to quantitate the fluorescence signals using sophisticated imaging software. Additionally, technical advances in fluorophore and microscope development have widened the selection of colors available for both single- and multi-color fluorescence imaging.

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Example of IHC detection by immunofluorescence (IF)
Fluorescence IHC detection of cytokeratin 18 in colon carcinoma tissue
Fluorescence IHC detection of cytokeratin 18 in colon carcinoma tissue. Human colon carcinoma sections were stained for cytokeratin 18 using an anti–cytokeratin 18 primary antibody (Cat. #MA5-12104) and Invitrogen DyLight 594–conjuated goat anti rabbit secondary antibody (Cat. #35560, red fluorescence). Thermo Scientific Pierce Hoechst stain was also used to fluorescently label cell nuclei (Cat. #62249, blue fluorescence).

Differences in fluorescence and chromogenic detection

Fluorophores are conjugated to secondary antibodies or probes like (strept)avidin in order to detect target antigens by IHC. Thermo Scientific NeutrAvidin Protein is a specially prepared form of avidin that decreases background biotin-binding. Each method of detection, chromogenic and fluorescence, has advantages and disadvantages. Key points to consider when deciding whether to use fluorescence or chromogenic IHC are discussed below.

Number of processing steps: A key difference between fluorescence and chromogenic detection methods is the number of steps in the staining protocol. Chromogenic detection requires the addition of a precipitating enzyme substrate at the end of the staining protocol. No signal is generated without this extra step. In contrast, fluorescence detection does not require any enzyme-mediated reactions that add additional steps to the protocol. Additionally, because enzymes are sensitive to neutralizing antibodies, pH, and buffer constituents; chromogenic detection usually requires more optimization.

Signal amplification: Both chromogenic and fluorescence-based IHC employ indirect methods to amplify the signal at specific antigenic sites. Detection is based on conjugating enzymes or fluorophores to secondary antibodies or to strept (avidin) or NeutrAvidin protein, which then binds to the biotinylated secondary antibody. Methods like the ABC method used in chromogenic IHC can form large avidin–biotin–enzyme complexes that greatly amplify the target signal over fluorescence methods. Other signal amplification strategies are possible.

Stability: Fluorophore-labeled tissue samples must be mounted with a solution containing an antifade compound to stabilize fluorescence. While the fluorescence may be detected for weeks to months after the slides are prepared and properly stored, only chromogenic methods permit long-term signal stability for years, in many cases.

Microscopy: While chromogenic methods of detection need only the simplest transmitted light microscope to view the IHC staining results, fluorescence detection methods require more expensive microscopes that provide fluorescence excitation at the correct wavelength and the appropriate emission filters for optimum multicolor imaging.

Image quality: Fluorescence detection methods provide better image quality for a number of reasons: 1) higher-resolution and multi-planar microscopy can be performed (i.e., confocal microscopy) with fluorescence microscopes, and 2) the precipitate formed by the chromogenic enzyme complex can cause "fuzziness" around the target antigen that prevents high resolution microscopy to determine protein localization.

Quantitation and high-throughput capabilities: In recent years, algorithms have been developed for the semi-quantitative analysis of chromogenic IHC, although the enzymatic nature of this approach prevents true quantitative results. This is in contrast to results obtained with fluorescent probes. In fact, the latest high-throughput approaches depend on fluorescence detection for rapid and quantitative automated microscopy (i.e., high-content screening).

Multiplexing: Multiple antigens can be labeled with different chromogens, although the antigens cannot be in close proximity because deposition of the first stain will mask the second antigen. Because fluorophores come in so many different colors, multiple antigens can be stained at the same time. This is enabled by conjugating multiple fluorophores to different primary antibodies or by using conjugated secondary antibodies to target primary antibodies derived from different species. This approach is ideal for high-resolution multi-antigen imaging in co-localization studies.

Learn more: IHC Troubleshooting Guide

Indirect fluorescence staining methods

Fluorophore-conjugated secondary antibody

This approach requires only a primary antibody and a fluorophore-conjugated secondary antibody, and is the simplest form of signal amplification. An added benefit of this approach is that multiple antigens can be labeled concurrently if the primary antibodies come rom separate species. The process is as follows:

  1. The primary antibody is incubated with the tissue sample to allow binding to the target antigen. Typical incubation times vary from 1 hour at ambient temperature to overnight at 4ºC.
  2. A fluorophore-conjugated secondary antibody, with specificity for the primary antibody, is incubated with the tissue sample to allow it to bind to the primary antibody. This incubation step usually lasts 1 hour at room temperature but can be extended to overnight at 4ºC.
  3. After some washing steps, the sample is then counterstained and mounted for microscopic visualization (imaging).

Fluorophore-conjugated strept(avidin)

This method offers greater amplification because of a 3- to 5-fold larger number of fluorophore molecules localized to the primary/secondary antibody complex. The process is as follows:

  1. The primary and biotinylated secondary antibodies are incubated with the tissue sample as indicated above.
  2. Fluorophore-conjugated (strept) avidin or NeutrAvidin protein is added to the tissue sample and incubated to allow all biotin-binding sites on the fluorophore-conjugated protein to be filled.
  3. The sample is then counterstained and mounted for microscopic visualization.

In this approach, multiple biotinylation events on each secondary antibody lead to multiple fluorophore-conjugated, biotin-binding proteins (avidin, streptavidin or NeutrAvidin). Each biotin-binding protein is conjugated to as many as 5 fluorophore molecules to yield the increased amplification.

Learn more: IHC Immunodetection

Recommended reading
  1. Coons AA et al. (1942) J Immunol  45:159–170
  2. Beisker W et al. (1987) Cytometry  8:235–239.
  3. Cowen T et al. (1985) Histochemistry  82:205–208.
  4. Mosiman VL et al. (1997) Cytometry  30:151–156.
  5. Romijn, Herms J et al. (1999) J Histochem Cytochem  47:229–236.

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