Immunostaining
By Palak Raisinghani | 3 September 2024
Immunostaining is a diagnostic process. It is used by pathologists to detect the presence of certain antigens present in sample of blood, bone marrow or tissue. It is widely used to diagnose infections, cancers, and other conditions.
Antigens are certain proteins or amino acid sequences, that are present on the surfaces of various body cells and foreign particulate objects entering the body. They are like markers telling the immune cells whether an object is foreign or not. The immune system generates antibodies highly specific for a certain antigen. They are shaped in such a way that they only lock on the antigens that they target. In simple words, an antigen is anything to which an antibody is generated.
The antigen has small sites on it which bind to the receptors of the antibodies - these are called epitopes. Epitoes are also referred to as antigenic determinants. These are the parts of antibodies that detect and bind to the epitopes of antigens.
The same principle of antigen-antibody interaction is used in immunostaining, except that here the antibodies are introduced into the sample externally (and not produced by the body). They bind to the target antigen only. The antibodies used in this process are linked to an enzyme or fluorophore. The conjugated enzymes are reacted further with a substrate, eventually forming a coloured or chemiluminescent product. Fluorophores are chemicals that absorb and emit energy in a predictable fashion. This eventually makes the antibodies easy to spot under the microscope. Pathologists interpret the slide and determine whether a target antigen is present or absent thus completing the diagnosis.
Immunostaining has emerged as a commonly used and valuable technique in diagnosis. However, some disadvantages are associated with the application of this technique. Immunostaining encompasses a few different methods:
- Immunohistochemistry: the antibodies bound to the antigens are stained with dyes or enzymes and this is placed on a glass slide that is evaluated under the microscope.
- Flow cytometry: the antibodies are stained, and a laser is used to analyses the sample and sort all the cells on by one into different categories.
- Immuno-electron microscopy: antibodies are linked to nanoparticles of gold. Gold is visible as dark flecks under an electron microscope; thus, it is easy to examine the sample.
Immunostaining methods are extensively used in protein purification, that is, a the process of separation of certain target protein from a mixture of other molecules/cells/tissues or whole organisms. Protein purification is a highly researched field as the process is often crucial in diagnosis of infections.
Immunostaining can be used to investigate the presence or absence of a certain protein, its distribution in a tissue and across regions and parts of a cell. Once the different proteins in a sample have been separated by SDS-PAGE and Western Blotting, the band corresponding to the protein is often identified using immunostaining. Antibodies against the protein of interest, conjugated with an enzyme or fluorophore (as before), are used for this purpose.
References
- Idleburg, C., Lorenz, M. R., DeLassus, E. N., Scheller, E. L., & Veis, D. J. (2021). Immunostaining of Skeletal Tissues. Methods in molecular biology (Clifton, N.J.), 2221, 261–273.
- Binch, A., Snuggs, J., & Le Maitre, C. L. (2020). Immunohistochemical analysis of protein expression in formalin fixed paraffin embedded human intervertebral disc tissues. JOR spine, 3(3), e1098.
- Maity, B., Sheff, D., & Fisher, R. A. (2013). Immunostaining: detection of signaling protein location in tissues, cells and subcellular compartments. Methods in cell biology, 113, 81–105.
- Image: BD Biosciences