Principles and advantages of immunohistochemical techniques



I. The basic principles of immunohistochemistry

Applying the principles of immunology and histochemistry, in situ qualitative, localization or quantitative research on certain chemical components in tissue sections or cell specimens, this technique is called immunohistochemical technique or immunocytochemical technique.

It is well known that the binding between antibodies and antigens is highly specific. Immunohistochemistry takes advantage of this feature, that is, to first extract certain chemical substances from tissues or cells, use them as antigens or haptens to immunize experimental animals such as mice, prepare specific antibodies, and then use the antibodies. (First antibody) as an antigen to immunize animals to prepare a second antibody, and then treat it with a certain enzyme (usually horseradish peroxidase) or biotin, and then combine with the aforementioned antigen components to amplify the antigen, because the antibody binds to the antigen The formed immune complex is colorless, therefore, the antigen-antibody reaction site must be displayed by means of histochemical methods (the commonly used chromogenic reagent DAB is shown as brown-yellow particles).

Through the antigen-antibody reaction and coloration reaction, the chemical components in the cells or tissues can be displayed, and the antigen-antibody reaction products in the cells can be clearly seen under the microscope, so that the distribution and content of certain chemical components can be determined in situ in the cells or tissues. . All substances in tissues or cells that can be used as antigens or haptens, such as proteins, polypeptides, amino acids, polysaccharides, phospholipids, receptors, enzymes, hormones, nucleic acids and pathogens, can be detected with corresponding specific antibodies.

II. 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., it can be divided into immunofluorescence, radioimmunoassay, Enzyme labeling method and immunogold and silver method, etc.

2. According to the dyeing steps, it 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 much improved.

3. According to the binding method, it can be divided into antigen-antibody binding, such as peroxidase-anti-peroxidase (PAP) method and affinity linkage, such as avidin-biotin-peroxidase complex (ABC) method, Streptavidin-peroxidase linkage (SP) method, etc., among which SP method is the most commonly used method.

III. Principles of several commonly used immunohistochemical methods

1. Immunofluorescence method

It is the earliest established immunohistochemical technique. It utilizes the principle of antigen-antibody specific binding, first label the known antibody with fluorescein as a probe to examine the corresponding antigen in cells or tissues, and observe it under a fluorescence microscope. When the fluorescein in the antigen-antibody complex is irradiated with excitation light, it will emit fluorescence of a certain wavelength, so that the localization of a certain antigen in the tissue can be determined, and further quantitative analysis can be performed. Due to its strong specificity, high sensitivity, rapidity and simplicity, immunofluorescence technology is widely used in clinical pathological diagnosis and testing.

2. Immunoenzyme labeling method

Immunoenzyme labeling is a technique developed in the 1960s after immunofluorescence. The basic principle is that the enzyme-labeled antibody interacts with tissues or cells first, and then the substrate of the enzyme is added to generate colored insoluble products or particles with a certain electron density. Antigen components for localization studies. Immunoenzyme labeling is the most commonly used technique at present.

Compared with the immunofluorescence technique, the main advantages of this method are: accurate positioning, good contrast, long-term preservation of stained specimens, and suitability for light and electron microscopy studies.

The development of immunoenzyme labeling methods is very rapid, and a variety of labeling methods have been derived. With the continuous improvement and innovation of methods, their specificity and sensitivity are constantly improving, and their use is becoming more and more convenient. At present, the widely used methods in pathological diagnosis are PAP method, ABC method, SP method and so on.

3. Immunocolloidal gold technology

The immunocolloidal 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 rapidly and stably, without obvious influence on the biological activity of proteins. Therefore, using colloidal gold-labeled primary antibodies, secondary antibodies or other molecules that can specifically bind to immunoglobulins (such as Staphylococcus protein A) as probes can be used to characterize, locate, and even quantify antigens in tissues or cells Research. 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. Because colloidal gold itself is pale to deep red, it is also suitable for light microscopy. For example, the silver-enhanced immune gold-silver rule is more convenient for light microscope observation.

IV. The advantages of immunohistochemistry

1. Strong specificity The basic principle of immunology determines that the binding between antigens and antibodies is highly specific. Therefore, immunohistochemistry is theoretically the specific display of antigens in tissue cells, such as keratin showing epithelial cells. composition, LCA showed lymphocyte composition. Cross-reactivity occurs only when cross-antigens are present in tissue cells.

2. High sensitivity In the initial stage of the application of immunohistochemistry, due to technical limitations, there were only direct and indirect methods with low sensitivity, and the antibodies at that time could only be diluted several times or dozens of times; now Due to the emergence of the ABC method or the SP method, the antibody can be diluted thousands of times, tens of thousands of times, or even hundreds of millions of times, and it can still bind to the antigen in the tissue cells. Such a highly sensitive antibody-antigen reaction makes the immunohistochemical method more and more It can be easily applied to routine pathological diagnosis work.

3. Accurate positioning, combination of shape and function This technology can accurately locate antigens in tissues and cells through antigen-antibody reaction and color reaction, so different antigens can be positioned and observed in the same tissue or cell at the same time. The study of the combination of morphology and function can be carried out, which is very meaningful for the in-depth study of pathology.