Fluorescence is emerging as a prime alternative to chromogenic approaches to immunochemistry (IHC) because of the ability to generate high-resolution images for protein localization studies and the capacity to quantitate the fluorescent signal. And technical advances in fluorophore and microscope development have widened the selection of colors to use for both single- and multi-color fluorescence microscopy greater than ever.

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Example of IHC Detection by Immunofluorescence (IF)

Fluorescent 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 and Thermo Scientific DyLight 594-Conjuated Goat Anti-Rabbit Secondary Antibody (red). Thermo Scientific Pierce Hoechst stain was also used to fluorescently label cell nuclei (blue).

Differences in Fluorescent and Chromogenic Detection

Fluorophores are conjugated to secondary antibodies or probes, such as avidin, to detect target antigens by IHC. Each method of detection--chromogenic and fluorescent--have legitimate benefitis and detractions, and listed below are key points to consider when deciding whether to use fluorescent or chromogenic IHC.

Number of processing steps: A key different between fluorescent and chromogenic detection methods is the number of steps to completion; chromogenic detection requires the addition of a substrate to develop the enzyme while fluorescent detection does not. Also, because enzymes are sensitive to neutralizing antibodies, pH and buffer constituents, more optimization is required for chromogenic detection.

Signal amplification: Both chromogenic and fluorescent IHC employ indirect methods to amplify the target antigenic signal by conjugating enzyme or fluorphores to secondary antibodies or avidin/streptavidin/NeutrAvidin Protein, which then binds to the biotinylated secondary antibody. But the ABC method used in chromogenic IHC can form large avidin-biotin-enzyme complexes that greatly amplify the target signal over fluorescent methods.

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 offer long-term stability of the signal for years.

Microscopy: While chromogenic methods of detection need only the simplest light microscope to view the target antigen, fluorescence detection methods require more expensive microscopes that provide fluorescence excitation at the correct wavelength.

Image quality: Fluorescent detection methods provide better image quality for a number of reasons: 1) higher-resolution and multi-planar microscopy can be performed (i.e., confocal) with fluorescent 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 semiquantitative analysis of chromogenic IHC, although the enzymatic nature of this approach prevents true quantitative capabilities, which can be performed 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 the first stain will mask the second antigen. Because of the myriad of fluorophore colors, multiple antigens can be stained at the same time, either through conjugation to different primary antibodies or by using conjugated secondary antibodies targeting primary antibodies from different species. This approach is ideal for high-resolution multiantigen imaging in colocalization studies.

Indirect Fluorescent Staining Methods

Fluorophore-conjugated Secondary Antibody

This approach requires only the primary antibody and a fluorphore-conjuated 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 are from separate species.

  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 against the primary antibody, is incubated with the tissue sample to allow binding to the primary antibody. This incubation step is usually 1 hour at room temperature but can be extended to overnight at 4ºC.
  3. The sample is then counterstained and mounted for microscopic visualization.

Fluorophore-conjugated Avidin

This method offers greater amplification because of a three- to five-fold greater number of fluorophore molecules localized to the primary/secondary antibody complex.

  1. The primary and biotinylated secondary antibodies are incubated with the tissue sample as indicated above.
  2. Fluorophore-conjugated avidin, streptavidin 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 recruits multiple fluorophore-conjugated biotin-binding proteins (avidin, streptavidin, or NeutrAvidin). Each biotin-binding protein is conjugated to as many as five fluorophore molecules to yield the increased amplification.

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