Mechanism And Principles Of Immunostaining

Immunostaining refers to the use of an antibody-based method of detecting a specific protein within a sample base. Albert Coons in the earlier part of the 20th century coined the term immnohistochemical staining, however, today, immunostaining refers to a broader range of techniques used in multi-disciplinary fields such as histology, cell biology and molecular biology, as long as the staining method uses an antibody as a means of detection.

The main principle is that we can visualize a target antigen by raising an antibody against it, therefore, if the target protein is present, a reaction will occur in the form of a “stain”, effectively, the antibodies in the serum used will bind to the target protein and generate a new “protein”/anti-“protein” complex. Alternatively, fluorescent dyes can be used – immunofluorescents.

Antibodies

There are two main types of antibodies which are used in research: monoclonal antibodies and polyclonal antibodies. The first contains a single antibody type that specifically recognizes a single epitope. Polyclonal antibodies are also produced in a similar manner as a monoclonal antibody, by injecting an antigen of interest into an animal (such as a rabbit) but the serum will contain a collection of antibodies that recognize different epitopes of the same protein.

The direct and indirect methods both refer to the detection of the required antibody which can then be visualized either with a fluorescent label or by using an enzyme such as peroxidase.

The Direct Method and Indirect Method

As the name would suggest, the direct method involves a labeled antibody reacting directly with the required protein in the sample of tissue section. Only one antibody is necessary to target the antigen in the tissue, which makes it a more straightforward technique. The direct method uses a polymer complex or a micro-polymer complex for immunostaining.

The indirect method involves multiple steps and the use of a labeled secondary antibody in addition to the (unlabeled) primary antibody, which would cause a reaction. This tends to be more sensitive than the direct method because of the signal amplification due to the binding that happens between the primary and secondary antibodies.

For an even stronger amplification, the secondary antibody can be conjugated to biotin molecules as well as the fluorescent or enzyme reporter. Biotin can bind with 3 different proteins: Avidin, Streptavidin and Neutravidin, causing a strong background staining due to additional enzyme activity and cross-reactivity. The short names for these are avidin-biotin complex (ABC) and streptavidin biotic complex (LSAB).

When to use direct and indirect methods

The selection of the best method depends on the level of target antigen expression, its accessibility and the type of readout desired. The Direct method is recommended to be used when trying to detect highly expressed antigens.

For most other applications, the indirect method tends to be preferred due to its ability to show a more powerful staining and the additional flexibility when choosing the secondary antibodies.

Controls

Fidelity can be assessed by using tissue controls:

• Positive control – section from the tissue which already expresses the target protein. It will confirm whether the reaction was successful through the fact that we already know that the protein of interest is expressed in the control tissue whereas it is unknown if it will be present in our chosen sample.
• Negative control – this is a similar example as above, only in this tissue we know with certainty that the protein is not expressed.
• Endogenous tissue background control – this is a section of the original tissue before the primary antibody was used on it.

Conclusion

Immunostaining refers to using an antibody based method to detect a targeted protein and is a technique which can be used both in diagnosis as well as research. Therefore, it exploits the principle of antibodies and gives opportunity for multiple ways of visualizing the results, leading to a wide variety of techniques used in molecular biology.