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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 diffferent species. This approach is ideal for high-resolution multiantigen imaging in colocalization studies.
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