Immunohistochemistry (IHC) is the application of the basic principle of immunology - the principle of antigen-antibody specific reaction, through chemical reaction to make the chromogenic agent (fluorescein, enzyme, metal ion, isotope) of the labeled antibody develop color to determine the antigen in tissue cells (polypeptides and proteins) for localization, qualitative and relative quantitative research. Immunohistochemistry has many advantages such as high specificity, high sensitivity, accurate positioning, combination of morphology and function. It can be applied to identify cell types, detect small metastases, understand the degree of differentiation, tumor origin and differentiation, and guide treatment and prognosis.
Immunohistochemistry Methods
Immunofluorescence Method
It is the earliest established immunohistochemical technique. It utilizes the principle of antigen-antibody specific binding, and first labels the known antibody with fluorescein as a probe to check the corresponding antigen in cells or tissues, and observes it under a fluorescent microscope. When the fluorescein in the antigen-antibody complex is irradiated by the excitation light, it will emit fluorescence of a certain wavelength, so that the location of a certain antigen in the tissue can be determined, and quantitative analysis can also be performed. Due to its strong specificity, high sensitivity, rapidity and simplicity, immunofluorescence technology is widely used in clinical pathological diagnosis and testing.
Immunoassay Method
The immunoenzyme labeling method is a technology developed in the 1960s after immunofluorescence. The basic principle is that enzyme-labeled antibodies are first interacted with tissues or cells and then enzymatic substrates are added to produce colored insoluble products or particles with a specific electron density, which can be studied by light or electron microscopy to localize various antigenic components on the cell surface and within the cells. This method is accurate in localization, has good contrast, stained specimens can be stored for long periods of time, and is suitable for light and electron microscopic studies, etc. The development of immunoassay methods has been very rapid. A variety of labeling methods have been derived, and their specificity and sensitivity are increasing as the methods continue to be improved and innovated. The ABC method, the SP three-step method, and the ready-to-use two-step detection system are now widely used in pathological diagnosis.
Immunocolloidal Gold Technology
The immunocolloidal gold technique uses colloidal gold, a special type of metal particle, as a marker. Colloidal gold is a hydrosol of gold, which rapidly and stably adsorbs proteins without any significant effect on the biological activity of the protein. Therefore, the use of colloidal gold labeling primary antibody, secondary antibody or other molecules that can specifically bind immunoglobulin (such as staphylococcal A protein) as a probe, can be qualitative, localization, or even quantitative study of the antigen in the tissue or cells. Because colloidal gold is available in different particle sizes and because of the high electron density of colloidal gold, the immunocolloidal gold technique is particularly suitable for single or multi-labeled localization studies in immunoelectron microscopy. As colloidal gold itself is pale to dark red in color, it is also suitable for light microscopic observation. Light microscopy is facilitated by the application of silver-enhanced immunogold and silver methods.
The Main Steps in the IHC
Frozen section preparation: It is recommended to use fresh tissue, otherwise the internal structure of the tissue cells will be destroyed, which will easily diffuse the antigen. Use clean and sharp blades, and the tissue must be properly frozen to prevent slivers and detachments.
Fixation of tissue slices: Immediately fix the cut slices with ice acetone or other fixative solution for 5-10 minutes after air-drying, especially white slices that need to be stored for a long time, must be fixed in time and properly stored.
Serum blocking: In order to prevent the binding of endogenous non-specific protein antigens, it is necessary to block with serum (same as the source of the secondary antibody) before the incubation of the primary antibody to weaken the background coloring. The time of serum blocking can be adjusted, usually 10-30min.
Primary antibody incubation conditions: The most important in immunohistochemical reactions, including incubation time and antibody concentration. There are several primary antibody incubation temperatures: 4°C, room temperature, and 37°C, among which 4°C is the best; incubation time: this is related to temperature and antibody concentration, generally 1-2 hours at 37°C, overnight at 4°C (after taking it out of the refrigerator rewarming at 37°C for 45 minutes).
Secondary antibody incubation conditions: Generally, at room temperature or 37°C for 30min-1h. In immunofluorescence, we generally set the secondary antibody concentration and incubation time first, and then explore the primary antibody concentration and incubation time.
Counterstaining: The purpose is to form a cell outline to better localize the target protein. DAPI counterstaining is commonly used.
Sealing: For long-term storage, we usually seal the slides with buffered glycerol, etc. In addition, there is a special anti-fluorescence extractive sealing solution. To avoid air bubbles, place a drop of sealing solution directly on the tissue of the slide, then hold one corner of the coverslip in one hand and the opposite corner in the other, lowering the corner close to the proximal end of the sealing solution until it touches the liquid, when you find that the liquid is diffusing on the contact surface, you can slowly lower the other corner, which usually does not produce air bubbles.
Washing of slices: In order to prevent non-specific staining caused by residual reagents such as primary antibodies and secondary antibodies, it is particularly important to properly strengthen washing (extend the time and increase the frequency). Washing after anti-incubation was performed 5 times × 5min.
Washing of sections: To prevent non-specific staining due to residual reagents such as primary and secondary antibodies, it is particularly important to intensify washing (longer and more frequent), generally 3 min × 3 times before incubation of primary antibodies and 5 times × 5 min after incubation of primary antibodies.
Visualization: The reporter molecule attached to the secondary antibody produces a visible signal, which can be detected using different techniques, such as enzyme-based chromogenic detection or fluorescence microscopy.
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