What is an immuno-histochemistry test?

Posted on  September 26, 2020, Edited by Jason, Category  

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What is immunohistochemistry?

1. Definition

Immunohistochemistry (ihc), also known as immunocytochemistry, refers to a new technology that the qualitative, localized, and quantitative determination of the corresponding antigens is performed through antigen-antibody reaction and histochemical color reaction in tissue cells’ situ by specific antibodies labeled with a chromogenic reagent.

It cleverly combines the specificity of the immune response and the visibility of histochemistry. With the help of the imaging and magnification of microscopes (including fluorescence microscopes and electron microscopes), it can detect various antigenic substances (such as protein, polypeptide, enzymes, hormones, pathogens and receptors, etc.). Immunohistochemistry technology has developed rapidly in recent years. In the 1950s, it was limited to immunofluorescence technology. And since then a highly sensitive and more practical immunoenzyme technology was gradually developed.

2. Rationale

The binding between antibody and antigen has a high degree of specificity, and immunohistochemistry harnesses this principle. Firstly, a certain chemical substance in the tissue or cell is extracted and used as an antigen or hapten. After immunizing the animal, a specific antibody is obtained, and then the antibody is used to detect the same antigen substance in the tissue or cell. Because the complex of antigen and antibody is colorless, it is necessary to show the binding site of antigen and antibody by means of histochemistry to achieve qualitative, localized or quantitative research on unknown antigens in tissues or cells.

3. Characteristics

A. Strong specificity

The basic principle of immunology determines that the binding between antigen and antibody is highly specific. Therefore, immunohistochemistry is theoretically a specific display of antigens in tissue cells. For example, keratin shows epithelial components, and LCA shows lymphocytes. ingredient. Only when there are cross-antigens in the tissue cells, cross-reactions will occur.

B. High sensitivity

In the initial stage of the application of immunohistochemistry, due to technical limitations, only the direct method, indirect method and other less sensitive technologies were available. At that time, the antibody could only be diluted for several times or dozens of times; now the advent of ABC method or SP allows antibodies to be diluted thousands, tens of thousands, or even hundreds of millions of times, and the antibody can still bind to antigens in tissue cells. This highly sensitive antibody-antigen reaction makes immunohistochemistry methods more and more convenient for routine pathological diagnosis.

C. Accurate positioning, combination of form and function

This technology can accurately locate antigens in tissues and cells through antigen-antibody reaction and color reaction, so that different antigens can be positioned and observed in the same tissue or cell at the same time. So the combination of morphology and function can be studied, which is very meaningful to carry out in-depth research in the field of pathology.

4. Classification

  • According to the types of labeling substances, such as fluorescent dyes, radioisotopes, enzymes (mainly horseradish peroxidase and alkaline phosphatase), ferritin, colloidal gold, etc., it can be divided into immunofluorescence, radioimmunoassay, immunoenzyme Labeling method and immune gold and silver method.
  • According to the dyeing steps, it can be divided into direct method (also called one-step method) and indirect method (two-step, three-step or multi-step method). Compared with the direct method, the sensitivity of the indirect method is much improved.
  • According to the binding method, it can be divided into antigen-antibody binding, such as peroxidase-antiperoxidase (PAP) method; affinity connection, such as avidin-biotin-peroxidase complex (ABC) method, chain Mold avidin-peroxidase linkage (SP) method, among which SP method is a more commonly used method; polymer linkage, such as a ready-to-use two-step method, which is especially suitable for high endogenous biotin content Tissue antigen detection.

5. Function

A. specimen

The experiments mainly use tissue specimens and cell specimens. The former includes paraffin embedding sections (pathological sections and tissue chips) and frozen sections, and the latter includes tissue prints, cell slides and cell smears.

Among them, paraffin section is the most commonly used and most basic method to make tissue specimens. It is well-preserved for tissue morphology and can be used for serial sectioning, which is conducive to various staining control observations; it can also be archived for a long time for retrospective research; It will have a certain impact on antigen exposure, but antigen retrieval can be performed, which is the preferred method of tissue specimen preparation in immunohistochemistry.

B. antibody

The commonly used antibodies in immunohistochemical experiments are monoclonal antibody and polyclonal antibody. Monoclonal antibodies are antibodies secreted by a B lymphocyte clone and are prepared by immunizing animals with cell fusion hybridoma technology. Polyclonal antibody is immune serum obtained from animal blood after the purified antigen is directly immunized. It is a mixture of antibodies produced by multiple B lymphocyte clones.

C. Common dyeing methods

According to the different markers, it is divided into immunofluorescence method, immunoenzyme labeling method, and affinity histochemistry method. The latter is a detection method based on a substance with a high affinity for a certain tissue component. This method is more sensitive and facilitates the localization of trace antigens (antibodies) at the cellular or subcellular level. Among them, the biotin-avidin staining method is the most commonly used.

What is immunohistochemistry used for?

Immunohistochemical staining has a very broad role in biomedical research and involves many research fields. However, immunohistochemistry technology also has its limitations. For example, the test substance in the tissue cells must be antigenic, and a certain concentration is required. The detected immunoreactive protein cannot be determined to be newly synthesized by the cell or transported through cells.

Therefore, these characteristics should be fully considered in experimental design. If the experiment needs to prove what kind of cell the known protein is synthesized by, molecular in situ hybridization technology is preferred. In order to guide beginners to use immunohistochemistry technology reasonably and skillfully in experimental design, the basic principles of its application are briefly described as follows:

  1. Determine the cell type and morphology. Some proteins in tissue cells have tissue specificity, such as glial fibrillary acidic protein (GFAP) only exists in astrocytes. Neurofilament (Neurofilament, NF) exists only in nerve cells. These tissue-specific proteins are usually referred to as labeled proteins (Proteiniliaker). The cell type can be determined by the specific antibody of the labeled protein.

    Some cells (such as Langerhans cells and melanocytes in the epidermis) are not easy to identify under a light microscope. By performing immunohistochemical staining on specific proteins in the cytoplasm, the outline of such cells can be clearly displayed. This effect is particularly necessary in neuroscience research and tumor clinicopathology.
  2. Identify the source of cell products. View certain cell products as antigens, prepare corresponding antibodies, and perform immunohistochemical staining on tissue cells to determine the source of cell products. For example, most of the hormones produced by endocrine cells can be identified by immunohistochemical staining techniques. Based on this, the secretory function of cells and the classification of endocrine tumors can be studied, and tumors that secrete ectopic hormones can be detected to understand the degree of cell differentiation.
  3. Determine the degree of cell differentiation. Different cells of the same type express different marker proteins, and the differentiation degree of the cells can be determined according to the identification of these different proteins. For example, the hallmark protein of neuroepithelial cells is nestin, which expresses vimentin when it differentiates into radial glial cells. In the neurogenesis phase, it expresses Ⅲβwhen it differentiates into neuroblasts neural tubulin (TUJl) and neurofilament (NF) when neuroblasts differentiate into mature neurons.
  4. Track the nerve fiber bundle and its projection area. The immunohistochemical method is often combined with the axoplasmic transport tracing method to study the connections between neurons. The axoplasmic transport tracing method uses certain substances that can be taken up by nerve endings. Use histochemical method to show the outline of neurons. Commonly used tracers include horseradish peroxidase and fluorescent gold.

    E.g. The aim is to observe the projection of nerve fibers from a nucleus in the peripheral nervous system or central nervous system. Firstly, the tracer is injected into the nerve fiber end of the animal to allow the animal to survive for a period of time. The material is taken from the expected nerve fiber projection site, and the tracer is located by the histochemical method, and then the immunohistochemical method is implemented to determine its nature.
  5. Application in clinical pathology. Such as identifying the nature of the lesion, discovering small lesions, exploring the origin or differentiated phenotype of the tumor, determining the tumor stage, guiding treatment and prognosis, assisting in the diagnosis and classification of diseases, and searching for the cause of infection, etc.

Immunohistochemitry Protocol

1. Immunofluorescence method

It is the earliest established immunohistochemistry technique. It uses the principle of antigen-antibody specific binding. First, the known antibody is labeled with fluorescein, which is used as a probe to check the corresponding antigen in the cell or tissue. If you observe it under a fluorescence microscope, it will emit fluorescence of a certain wavelength when the fluorescein in the antigen-antibody complex is irradiated by the excitation light, it. Then the localization of a certain antigen in the tissue can be determined, and quantitative analysis can be performed. Because of its strong specificity, high sensitivity, quickness and simplicity, immunofluorescence technology is widely used in clinical pathological diagnosis and testing.


  • Application in autoimmune diseases
  • Rapid identification of bacteria and viruses
  • Detection and research of parasites

2. Immunoenzyme labeling method

The immunoenzyme labeling method is a technique developed in the 1960s followed by immunofluorescence. The basic principle is to first use enzyme-labeled antibodies to interact with tissues or cells, and then add enzyme substrates to generate colored insoluble products or particles with a certain electron density.

Through light or electron microscopy, various types of cell surfaces and antigen component in cells is subject to localization research. Immunoenzyme labeling technology is the most commonly used technology. The main advantages of this method compared with immunofluorescence technology are: accurate positioning, good contrast, long-term preservation of stained specimens, and suitable for optical and electron microscopy studies.

The immunoenzyme labeling method has developed very rapidly, and a variety of labeling methods have been derived. With the continuous improvement and innovation of the method, its specificity and sensitivity have been greatly improved, more convenient to use. ABC's method, SP three-step method, ready-to-use two-step method detection systems are widely used in pathological diagnosis.


  • Improve the accuracy of pathological diagnosis
  • Clinical application of oncogene protein
  • Evaluation of the degree of tumor cell proliferation
  • Discovery of micrometastases
  • Significance in tumor staging
  • Guide the treatment of tumors
  • Assist in the diagnosis of immune diseases and infectious diseases

3. Immune colloidal gold technology

The immune colloidal gold technology uses a special metal particle such as colloidal gold as a marker. Colloidal gold refers to the hydrosol of gold, which can adsorb proteins quickly and stably with no obvious effect on the biological activity of the protein. Therefore, using colloidal gold-labeled primary antibody, secondary antibody, or other molecules that specifically bind to immunoglobulins (such as staphylococcal protein A) as probes, can qualitatively locate, or even quantify antigens in tissues or cells.

Since colloidal gold has particles of different sizes and the electron density of colloidal gold is high, the immuno-colloidal gold technique is particularly suitable for single-label or multi-label localization studies of immunoelectron microscopy. And because of the color of colloidal gold from light red to deep red, it is also suitable for light microscope observation. Such as the application of silver-enhanced immunogold and silver law is more convenient for light microscope observation.


Because of its own unique characteristics, immunohistochemistry technology has become a technique often selected by the majority of scientific researchers in tissue cell positioning, qualitative and quantitative research. Therefore, with the update of scientific research tools, scientists need to have a more extensive and in-depth understanding of immunohistochemistry, optimize the design of technical routes, and promote the further development of immunohistochemistry technology.

Want to learn more about antigens and antibodies? Read about FC stand information.


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