Immobilization of antigens in tissue cells Immobilization of antigens in tissue cells is an important part of immunoenzymatic and non-labeled immunoenzyme histochemical methods. After fixation, the following purposes can be achieved: 1. The attraction of the specimen on the slide is increased; 2. The lipid and waxy components contained in the specimen can be removed, which is conducive to the combination of antigen and enzyme-labeled antibody; 3. The specimen The storage time can be increased. After the tissue section is fixed, it can be stored at -20 ℃ for more than one year without changing the staining of the immunoenzyme technology; if the relevant antigens in the tissue are different, the fixing agent and fixing method used will also change. Acetone and ethanol are two commonly used fixatives. However, for the location studies of many viruses and bacteria, it is most appropriate to choose acetone and carbon tetrachloride as fixatives; for the location of soluble protein antigens, ethanol is commonly used; for polysaccharides, 8% -10% formalin is used. Instead of using organic solvents such as ethanol and acetone. For those antigens covered with protein coating on the surface, trypsin, mucin, hyaluronidase, and receptor-damaging enzymes are also commonly used. It has been reported that the application of picric acid-formaldehyde fixative solution in the positioning of alpha-fetoproteinase in liver tissue can achieve satisfactory results. For the fixation of plant virus materials, acetone is used most, followed by ethanol. Because the acetone has minimal damage to the virus, but for those unstable viruses, you should avoid fixation. When applying immunoenzyme technology, the effect of fixation treatment on the activity of the enzyme is also well known. Many fixatives can impair the activity of antigens. For example, formalin fixation of protein antigens causes the destruction of antigenicity is very obvious. Therefore, the concentration of formalin must be strictly controlled. The formalin concentration generally used is 10%. Nevertheless, the antigenicity of the antigen is still damaged, so antigen repair must be performed. In addition, for alkaline phosphatase, at 4 ° C, acetone fixation throughout the day and night can lose 30% of the activity. If embedded with acetone, it loses 70% of the activity; at 4 ° C, formalin fixation 2 ï¼4h, it loses 25% of its activity. The fixing conditions have a greater effect on the fixing effect of the specimen. Fixative concentration: acetone is most commonly used at 100%; ethanol is most commonly used at 95%, but 70% ethanol is the strongest at fixation; formalin is commonly used at 10%. Fixed temperature: Generally speaking, the fixing effect strengthens with increasing temperature, and room temperature is the most commonly used. The fixed temperature can be -70-30 ℃. Different temperatures should be used according to different antigens. The measles virus should be fixed with acetone at -20-40 ° C for 30min, and the enzyme can be fixed with ethanol at 37 ° C for 15min. If an enzyme is used to remove the protein coating on the surface of certain antigens, the pH should be adjusted according to the nature of the enzyme used. Dry humidity when fixed: Dry humidity does not generally need to be considered, but certain materials are greatly affected by dry humidity when fixed. Acetone fixes the poliovirus in Hela cells. When dried at room temperature, the N antigen changes to H antigen; while it does not dry at -20 ℃, the N antigen remains unchanged. Fixing time: long and short, usually at room temperature, fixed for about 15min, fixed at low temperature for 30min. If only to prevent the material from falling off in order to reduce the cavitation of untreated fresh frozen sections, acetone, ethanol and formaldehyde can be used Do it for a short time. Rinse after fixation: The material after fixation is usually washed repeatedly with neutral phosphate buffer solution, so as not to cover the surface with substances that may be diffused during the fixation process. Preservation of fixed specimens: After the specimen materials are fixed, they should be used in immunoenzyme technology immediately, and should not be stored for a long time. If it is really to be stored, it should be placed in a refrigerator (4 ℃), and the antigenicity will not be lost within 24 hours under dry conditions. At -20 ℃, frozen sections or smears can be stored for 1 week to 1 month, and paraffin sections can be stored for more than several months. When stored at -20 ° C, in order to prevent the formation of excessive ice crystals and changes in tissue cells, the sections should be immersed in the solution of polyvinyl alcohol and glycerin, and allowed to form a solid protective film at -20 ° C, and then immune Before staining, warm with fingers and wash off gently with physiological saline, 5 minutes each time, a total of 3 times. The specimens preserved in this way have increased pollution. Certain dry specimens (such as amoebic antigens in tissues and tissue self-antigens) can be stored in octane for long-term storage (-30 ° C) and are almost free of contamination. The fixation procedure is very simple, add fixative to the specimen with a pipette, or place the specimen on the slide rack and immerse in the fixative solution, rinse with phosphate buffer immediately after fixation, and then dry in the air or blow dry with a fan .
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