IHC Staining Methods

Various IHC Staining Methods

You can use a variety of methods to stain IHC samples for detection. Of course, they all relate to the use of antibodies for specific detection, you will find that certain methods are better than others in different applications. Here is a short list and description for different detection methods available in IHC.

Peroxidase-based Methodology

One of the most common detection methods at our disposal is horseradish peroxidase coupled to a chromogenic substrate. This is still in widespread use today, and it was actually one of the first IHC methods developed.

Avidin-Biotin Affinity

Biotin is a vitamin with strong affinity for the streptavidin protein. Peroxidase molecules are tagged with streptavidin and then are used to bind biotin-labeled secondary antibodies. This facilitates rapid and selective imaging of the antibody binding sites. An improvement on the method using streptavidin molecules labeled with multiple detectors was developed later to improve signals.

Unfortunately, this method can easily be disrupted by the presence of endogenous biotin in different tissues. Antigen retrieval in particular can promote this issue.

Polymer Affinity

An alternative to the biotin detection system is the coupling of multiple antibodies and detection enzymes to one polymer molecule. This introduces a one-step system. The antibody will locate the polymer scaffold in the vicinity of the protein of interest. Then the detection enzymes can be used to generate a large signal. Unfortunately, this method requires special synthesis of specific antibodies with the polymer. Therefore, it is not particularly versatile.


Human tonsil tissue sections stain with mouse anti-Ki67
primary antibody and detected using GeneTex
OneStep Polymer (incubation time, 15 minutes at RT).

An alternative polymer method couples secondary antibodies to a scaffold containing the detection enzymes. This creates a universal detection reagent useful for the analysis of a wide variety of different primary antibodies.

Tyramide Amplification and Derivatives

Tyramide molecules can be forced to form chemical bonds with peroxidase molecules. When biotin is conjugated to the tyramide, you create a system where one peroxidase molecule is coupled to many biotins. If you then incubate using streptavidin-peroxidase, you will amplify the signal immensely because one secondary antibody will have far more than one peroxidase molecule associated with it. This can be repeated a few times to exponentially increase the signal, but eventually the background of the signal will be rendered too high to continue.

An alternative tyramide method is to couple the molecule to a fluorescent dye instead of biotin. In this case, the many tyramide molecules attached to peroxidase can be visualized as a fluorescent signal. This saves time and money on the incubation steps required for traditional tyramide approaches.

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