Chromogenic detection relies on enzymes that convert soluble substrates into insoluble chromogenic products. Alternatively, fluorescent detection uses fluorochrome labels, which emit light of a longer wavelength when excited by light of a specific wavelength.

Chromogenic detection in IHC

The enzymes used to detect antigens in chromogenic detection are typically conjugated to secondary antibodies, although primary antibodies directly conjugated to the enzyme can also be used.

The most commonly used enzymes are horseradish peroxidase (HRP), which converts 3,3'-diaminobenzidine (DAB) into a brown product, and alkaline phosphatase (AP), which converts 3-amino-9-ethyl carbazole (AEC) into a red product. However, the choice of chromogen is determined by the enzyme used.

Table 4. View guidelines for choosing which chromogen to use

Four principal methods of indirect chromogenic detection are widely used. The biotin-based methods use an avidin-biotin complex (ABC) or a labeled streptavidin-biotin binding (LSAB) complex. The non-biotin based methods employ a polymer complex or a micropolymer complex

Avidin-biotin complex (ABC) method

Early use of the ABC method relied on biotinylated secondary antibodies and an avidin-biotin- reporter enzyme complex. As avidin is tetravalent, large complexes form, resulting in high signal intensity.

Figure 3. Antibody detection via avidin-biotin complex method

Labeled streptavidin-biotin (LSAB) method

Most detection now relies on the LSAB variant of the ABC method, which uses streptavidin, instead of avidin. This results in less non-specific tissue binding, as streptavidin is not glycosylated and has a more neutral isoelectric point than avidin.

Figure 4. Antibody detection via streptavidin-biotin method

Polymer methods

The key challenge of biotin-based systems is that endogenous biotin can lead to significant background staining in certain tissues (eg brain). While formalin fixation and paraffin embedding reduce biotin levels, antigen retrieval can expose biotin.

In frozen sections, endogenous biotin is a significant problem. Although extra steps with biotin blocking solutions can be used to reduce background, non-biotin polymer-based methods offer an alternative.

Early polymer methods used a dextran backbone to which multiple enzyme molecules and secondary antibodies are attached. More recent micro-polymer / compact polymer methods use a smaller detection complex with less tendency to aggregate. This results in greater sensitivity through better tissue penetration and reduced background staining from endogenous biotin.

Figure 5. Antibody detection via dextran backbone

Counterstains and special stains for IHC

Histology, fluorescent, and special stains are used to stain specific cell and tissue structures and microorganisms to help identify the location of primary antibody staining.

Chromogenic counterstains  are used when the primary antibody is visualized using HRP or alkaline phosphatase combined with DAB, AEC, or similar enzyme substrates. The traditional combination is HRP/DAB with hematoxylin to stain nuclei. Eosin, for cytoplasmic staining, is often used in combination with hematoxylin.

Fluorescent counterstains are used when the primary antibody staining is visualized with a fluorescent dye conjugated directly to the primary or secondary antibody. You should avoid using a counterstain with an overlapping emission spectrum to the dye used for primary antibody visualization. DAPI is traditionally the most popular fluorescent nuclear counterstain.

Special stains are used to stain cell types, microorganisms, and specific proteins, carbohydrates, and metabolites found in the tissue matrix and within cells. While simple to use once established, special stains are often time-consuming to set up and optimize. Optimized special stain kits provide a convenient alternative to setting up a stain yourself, and they are often faster to use than the traditional staining methods.

Figure 5. Common counterstains and their targets

Figure 6. Example of the use of special stains and counterstains. Prussian blue iron stain kit staining iron (blue) in liver tissue. Nuclei and background were counterstained with nuclear fast red.

Advantages of chromogenic detection over fluorescent detection

Chromogenic detection is usually more sensitive than fluorescent detection due to the higher signal amplification. Furthermore, unlike fluorophores, the colored precipitates created from substrates such as DAB are photostable, enabling storage of the slides for many years.

While fluorescent detection requires specialized light sources and filters, chromogenic detection only requires a standard microscope. However, the experimental procedure is longer as it includes more incubation and blocking steps than fluorescent methods.

Multicolor and fluorescent detection in IHC

Staining up to three antigens simultaneously using chromogens is possible, which typically requires primary antibodies raised in different species. If the antigen is present at a high enough level, a primary antibody directly conjugated to the reporter enzyme can be used. If you are using two primary antibodies raised in the same species, binding sites on the secondary antibody must be blocked before staining with the second primary antibody.

Alternatively, fluorescent detection is widely used to visualize multiple antigens simultaneously. The fluorochrome may be conjugated directly to the primary or secondary antibody or streptavidin.

When designing multicolor experiments, two key parameters must be considered:

• The spectral overlap between the fluorochromes being used should be limited as much as possible.

• If indirect detection methods are employed, cross-reactivity between the detection reagents must be avoided where possible. This is usually achieved by selecting primary antibodies from different species, ensuring that each secondary antibody only recognizes one primary antibody in the experiment.

Two primary antibodies from the same species may be used if one of the primary antibodies is biotinylated. The tissue is first incubated with the non-biotinylated antibody and the corresponding fluorochrome-conjugated secondary antibody. The tissue is then incubated with the biotinylated antibody, followed by a streptavidin-conjugated fluorophore. This method is susceptible to high background staining due to endogenous biotin, particularly when using frozen tissues

Mounting media

After staining, a mounting medium is used to adhere a coverslip to a tissue section or cell smear. Mounting the tissue specimen is essential for preservation during storage and enhancing imaging quality during microscopy.

There are two categories of mounting media, organic and aqueous (or hydrophobic and hydrophilic, respectively). Organic mounting media can only be used for enzymatic labels where the precipitate formed between the enzyme and the chromogen is not soluble in the organic solvents used during mounting of the tissue (eg diaminobenzidine, DAB).

Aqueous mounting media are generally suitable for all enzymatic label/chromogen combinations and fluorescent labels. If you are using fluorescent labels, use a mounting media that preserves fluorescence to enable imaging of samples after prolonged storage.

Recommended mounting media for non-fluorescent imaging

Recommended mounting media for fluorescent imaging