Immunohistochemistry

Immunohistochemistry (IHC), also known as immunohistology, describes the staining of proteins and cell or tissue structures using dye-coupled antibodies [1]. When this involves a fluorescent dye, it is also referred to as immunofluorescence [2]. Since the goal is to detect the signal only in the area of the epitope, the binding to the antibody should be very strong and specific. The evaluation of the color pattern and its intensity is usually performed with the help of a light or fluorescence microscope [3]. A distinction is made between different staining methods.

Direct Staining

In direct staining, a primary antibody is coupled with the corresponding detection system. That is, the antibody that binds the epitope is labeled with a dye. This method is well suited for imaging several different antigens in a sample. However, the color signal may not be sufficient if only a small amount of antigen is present in the specimen [4].

Indirect Staining

In indirect staining, the epitope is recognized by an unlabeled primary antibody. In addition, a secondary antibody is used, which binds to the primary antibody. This second antibody leads to a signal amplifying effect, so that stronger signals can be achieved even with lower antigen amounts. In addition, this variant is less expensive than the direct method, which is why it is used more frequently in routine immunohistological laboratories [4].

Enzyme-based Staining

In addition to the aforementioned detection by a fluorophore, enzymes are often used in IHC. In these so-called chromogenic indicators, an enzyme reacts with a substrate to produce an intense color signal that can be analyzed with an ordinary light microscope. The enzymes alkaline phosphatase (AP) and horseradish peroxidase (HRP) are most commonly used for antigen detection. They convert a number of chromogenic, fluorogenic, and chemiluminescent substrates, including DAB (3,3'-diaminobenzidine), producing a brown staining [2].

Procedure of an immunohistochemical assay. In the first step, a sample is taken from the tissue to be examined, with which an immunohistochemical assay is performed: First, a specific primary antibody binds to the target antigen from the tissue sample. Subsequently, a labeled secondary antibody is used, which binds to the primary antibody and catalyzes a color reaction after addition of the corresponding substrate. The resulting color pattern can then be evaluated under a microscope (figure created with biorender.com).

To begin immunohistochemical examinations, the specimen is first prepared by fixing the tissue, embedding it in kerosene, and sectioning it with a microtome. Since the antigen structure may be altered by this pretreatment, the important step of antigen retrieval follows to break up the protein linkages formed during fixation and to unmask the antigen binding sites. The most common methods of recovery are heat-induced epitope retrieval (HIER), which uses a heating source together with buffers and enzymes, and protease-induced epitope retrieval (PIER), which uses various proteases such as proteinase K, trypsin or pepsin [5].

IHC is particularly used in medical histology to detect and classify tumor cells on the basis of specific antigens. In this way, malignant tissue can be differentiated from benign tissue. Thus, IHC supports tumor diagnostics in prognosis and prediction of response to certain therapies. Furthermore, it simplifies research of the origin and growth behavior of tumors. In addition to cancer diagnostics, immunohistochemistry is also used to detect pathological deposits and infectious agents such as herpes or hepatitis viruses [6].

Are you looking for the right antibody for your next immunohistochemical examination? You can be sure to find it at Biomol! We offer a wide selection of both primary and secondary antibodies:

Primary Antibodies for Immunohistochemistry at Biomol

Secondary Antibodies for Immunohistochemistry at Biomol

Sources

[1] Gudrun Lang: Histotechnik. Springer-Verlag. S. 271 (2013).

[2] https://en.wikipedia.org/wiki/Immunohistochemistry, 24.01.2023

[3] https://flexikon.doccheck.com/de/Immunhistochemie, 24.01.2023

[4] Chen X, Cho DB, Yang PC. Double staining immunohistochemistry. N Am J Med Sci. 2(5): 241-5 (2010).

[5] McClellan P, Jacquet R, Yu Q, Landis WJ. A Method for the Immunohistochemical Identification and Localization of Osterix in Periosteum-Wrapped Constructs for Tissue Engineering of Bone. J Histochem Cytochem. 65(7): 407-420 (2017).

[6] https://ipa.med.ovgu.de/Krankenversorgung/Klinische+Pathologie/Immunhistologie.html, 24.01.2023