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Principles, classification and advantages of immunohistochemistry
First, the basic principles of immunohistochemistry technology using immunology and histochemistry principles, in situ qualitative, localized or quantitative study of certain chemical components in tissue sections or cell specimens, this technique is called immunohistochemistry or Immunocytochemistry technology. It is well known that the binding between an antibody and an antigen is highly specific. Immunohistochemistry utilizes this feature by first extracting certain chemicals from tissues or cells and using them as antigens or haptens to immunize experimental animals such as mice to prepare specific antibodies. (First antibody) is used as an antigen to immunize an animal to prepare a second antibody, and is treated with an enzyme (usually horseradish peroxidase) or biotin, and then combined with the aforementioned antigen component to amplify the antigen, since the antibody binds to the antigen. The immunocomplex formed later is colorless, and therefore, the antigen-antibody reaction site must also be displayed by means of histochemical methods (the usual developer DAB is shown as brownish yellow particles). Through antigen-antibody reaction and color reaction, the chemical components in cells or tissues are displayed. Under the microscope, the antigen-antibody reaction products occurring in the cells can be clearly seen, so that the distribution and content of certain chemical components can be determined in situ in cells or tissues. . Any substance or antigen that can be used as an antigen or hapten in a tissue or cell, such as a protein, a polypeptide, an amino acid, a polysaccharide, a phospholipid, a receptor, an enzyme, a hormone, a nucleic acid, and a pathogen, can be detected by a corresponding specific antibody. Second, immunohistochemical staining method 1, according to the type of labeling substances, such as fluorescent dyes, radioisotopes, enzymes (mainly horseradish peroxidase and alkaline phosphatase), ferritin, colloidal gold, etc., can be divided into immunity Fluorescence, radioimmunoassay, enzyme labeling, and immunogold and silver methods. 2, according to the dyeing step can be divided into direct method (also known as 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 improved a lot. 3, according to the combination of antigen-antibody binding, such as peroxidase-anti-peroxidase (PAP) method and affinity linkage, such as avidin-biotin-peroxidase complex (ABC) method, The streptavidin-peroxidase-linked (SP) method and the like, among which the SP method is the most commonly used method. Third, the principle of several commonly used immunohistochemical methods 1, immunofluorescence method is the earliest established immunohistochemistry technology. It uses the principle of antigen-antibody specific binding, first labeled known antibodies with fluorescein, as a probe to examine the corresponding antigen in cells or tissues, and observed under a fluorescence microscope. When the fluorescein in the antigen-antibody complex is irradiated by the excitation light, a certain wavelength of fluorescence is emitted, thereby determining the localization of an antigen in the tissue, and further quantitative analysis. Because of its strong specificity, high sensitivity, fast and simple, immunofluorescence technology is widely used in clinical pathological diagnosis and testing. 2. Immunoenzymatic labeling method The immunoenzymatic labeling method is a technique developed in the 1960s following immunofluorescence. The basic principle is that the enzyme-labeled antibody acts on the tissue or cells, and then the substrate of the enzyme is added to produce a colored insoluble product or a particle having a certain electron density, and the cell surface and the intracellular cells are irradiated by light or electron microscopy. The antigen component was subjected to localization studies. Immunolabeling technology is currently 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, suitable for light and electron microscope research. The development of immunolabeling methods has been very rapid, and a variety of labeling methods have been derived. With the continuous improvement and innovation of the methods, the specificity and sensitivity are continuously improved, and the use is more and more convenient. Currently widely used in pathological diagnosis are PAP method, ABC method, SP method and the like. 3. Immunocolloidal gold technology The immunocolloidal gold technology uses a special metal particle such as colloidal gold as a marker. Colloidal gold refers to a gold hydrosol that adsorbs proteins quickly and steadily without a significant effect on the biological activity of the protein. Therefore, using a colloidal gold-labeled primary antibody, secondary antibody, or other molecule that specifically binds to an immunoglobulin (such as Staphylococcal Protein A) can be used as a probe to characterize, localize, or even quantify antigens in tissues or cells. the study. Because colloidal gold has particles of different sizes and the electron density of colloidal gold is high, the immunocolloidal gold technique is particularly suitable for single-label or multi-label localization studies of immunoelectron microscopy. Since colloidal gold itself is light to deep red, it is also suitable for light microscopy. For example, the use of silver-enhanced immunogold and silver rule is more convenient for light microscopy. Advantages of immunohistochemical technique 1. The basic principle of specific strong immunology determines that the binding between antigen and antibody is highly specific. Therefore, immunohistochemistry is theoretically also a specific display of antigen in tissue cells. For example, keratin shows the epithelial component and LCA shows the lymphocyte component. Cross-reactivity occurs only when cross-antigens are present in tissue cells. 2, high sensitivity in the initial stage of application of immunohistochemistry, due to technical limitations, only direct methods, indirect methods and other low-sensitivity technology, then antibodies can only be diluted several times, dozens of times; Due to the appearance of the ABC method or the SP method, the antibody can be diluted with thousands of times, tens of thousands of times or even hundreds of millions of times to bind to the antigen in the tissue cells, so that the highly sensitive antibody antigen reacts to make the immunohistochemical method more and more Conveniently applied to routine pathological diagnosis work. 3, accurate positioning, morphology and function combined with the technology through the antigen-antibody reaction and color reaction, the antigen can be accurately located in the tissue and cells, so that different antigens can be positioned in the same tissue or cells at the same time, so It is possible to conduct a combination of morphology and function, which is very meaningful for the depth of pathology research.