1. The key links of enzyme immunohistochemistry 1. Specimen fixation: The purpose of fixation is to ①prevent the specimen from falling off from the slide; ② remove the lipid that prevents the antigen-antibody binding, so that the antigen-antibody conjugate is easy to obtain good staining results; ③The fixed specimen is easy to save. 2. Dehydration, paraffin embedding and tablet production: Gradient ethanol (from low to high) for dehydration is fully dehydrated, the tissue must be completely immersed in wax, and the blade should be clean and sharp when slicing. Otherwise, it is easy to split and take off. 3. Dewaxing and hydration: This is because the subsequent antibodies and other reagents can fully react with the antigens in the tissue. Dewaxing can be done at 60 degrees for 20 minutes, then immediately xylene 1-3 for 10 minutes, but the slices made on the same day can be 60 degrees for 3-4 hours. Gradient ethanol for hydration (from high to low). If dewaxing and insufficiency of hydration are prone to focal reactions and incomplete immersion, non-specific background coloration occurs. 4. Antigen repair: As part of the antigen in the tissue is fixed by formaldehyde or paraformaldehyde, cross-linking between proteins and the sealing effect of aldehyde groups occur, thereby losing antigenicity; through antigen repair, the intracellular antigenic determinants are re-established Exposure, improve antigen detection rate. Commonly used repair methods are generally divided into three types from strong to weak, high-pressure repair, microwave repair, pancreatin repair. There are also several types of repair fluids (specific information can be found, a large number: neutral, high pH, ​​etc.). Our laboratory generally uses microwave to repair medium heat for 6min * 4 times, and the effect is good. Note that after microwave repair, it will naturally cool for about 30 minutes (as long as you think the temperature of the repair solution reaches room temperature). 5. Cell permeability: The purpose is to allow the antibody to fully enter the cell for the binding reaction. Permeate solutions such as Triton X-100 and proteinase k are generally used. For example, Triton X-100 can dissolve the lipids on the cell membrane, nuclear membrane, and organelle membrane to allow antibodies and macromolecular structures to enter the cytoplasm and nucleus, so it is particularly recommended for cellular immunohistochemistry, so that the antibody can Smoothly enter the cell and bind with the corresponding antigen. In immunohistochemistry (> 10um thick sections) and immunocytochemistry, Triton X-100 is generally used as a cell permeating agent, and the membrane is perforated. 6. Inactivation of endogenous peroxidase and biotin: In the traditional ABC method and SP method, the results of immunohistochemical reactions are susceptible to the interference of endogenous peroxidase and biotin, and peroxide must be used Hydrogen and avidin are inactivated. The inactivation of endogenous POD is generally 3% hydrogen peroxide. The inactivation time is short, which can be about 10 minutes, and 0.3% hydrogen peroxide can appropriately extend the closing time, generally 10 to 30 minutes; use methanol to configure hydrogen peroxide than double Distilled water or PBS may play a role in protecting the antigen and fixing the tissue. Hydrogen peroxide incubation for too long may easily cause off-chips; use it now, and store it in the dark at 4 degrees. However, there is now a "second-generation ready-to-use immunohistochemistry kit" to avoid the interference of endogenous biotin, which is recommended. 7. Serum blocking: There are remaining sites on the tissue section that can non-specifically bind to the primary antibody, resulting in false positives for subsequent results; the blocking serum is generally from the same source as the secondary antibody. There are cross-reactive sites in the combination; calf serum, BSA, sheep serum, etc. can also be used, but they cannot be consistent with the source of the primary antibody. Generally room temperature or 37 degrees for 10-30min. 8. Primary antibody and secondary antibody concentration and incubation time: The primary antibody incubation conditions are the most important in the immunohistochemical reaction, including incubation time and antibody concentration. There are several kinds of primary antibody incubation temperature: 4 degrees, room temperature, 37 degrees, of which 4 degrees is the best; incubation time: this is related to temperature and antibody concentration, generally 37 degrees 1-2h, and 4 degrees overnight and taken out from the refrigerator After 37 degrees rewarming 45min. The specific conditions have to be explored. Secondary antibody incubation conditions: the secondary antibody is usually at room temperature or 37 degrees for 30min-1h, the specific time needs to be explored, and the concentration generally has a working solution, and if the concentrated solution is to explore the concentration. However, in immunohistochemistry, we generally set the concentration of the secondary antibody and the incubation time first, and then explore the concentration and incubation time of the primary antibody. 9. Antibody diluent: In fact, many laboratory antibody diluents can use general PBS, but in addition to the PBS component, the special antibody diluent also includes sodium azide preservative, BSA stabilizer and other components. Multiple antibody recycling is better. It is for this reason that I have been using domestic-made special antibody dilutions. For a period of time, when the new antibody dilutions were replaced, the experimental results showed negative results (prompting that the primary antibody may not be bound). Finally, from the antibody concentration and incubation time, blocking time After excluding the reasons, it was found that the pH value of the new antibody dilution was acidic, which caused the antigen-antibody reaction to be poor, and eventually a false negative result appeared. 10. Section cleaning (dipping, rinsing and rinsing): In order to prevent the non-specific staining caused by the residual of the primary antibody and secondary antibody, it is particularly important to strengthen the cleaning (extend the time and increase the number of times). I generally incubate with the primary antibody The previous cleaning is 3min * 3 times, and the cleaning after the primary antibody incubation is 5 times * 5min. Note: (1) Rinse separately to prevent contamination caused by cross-reaction. (2) Rinse gently to prevent the slice from falling off. I like to use the dip method; (3) The rinsing time should be enough to thoroughly remove the combined substances. (4) Use and requirements of PH and ionic strength of PBS. I have a painful lesson in this regard. At that time, the antibody dilution I bought was slightly acidic, and the result was a yellow background (no specific staining was seen). It is recommended that the pH is 7.4-7.6 and the concentration is 0.01M. (Neutral and weak alkaline conditions (PH7-8) are conducive to the formation of immune complexes, while acidic conditions are conducive to decomposition; low ionic strength is conducive to the formation of immune complexes, and high ionic strength is conducive to decomposition) 11 2. DAB color development: the depth of background and the depth of specific staining can be determined by DAB incubation conditions. The color development time of DAB is not fixed. The color development time is mainly controlled by the microscope, and it can be rinsed when the specific staining is strong and the background coloring is light; DAB color development time is very short (such as a few seconds or tens of seconds) There is a dark brown color, which may indicate that your antibody concentration is too high or the antibody incubation time is too long, you need to lower the antibody concentration or shorten your antibody incubation time; In addition, if the background appears very dark for a short time, also It is possible that the blocking non-specific protein in front of you is incomplete and you need to extend the blocking time; DAB coloring time is very long (such as more than ten minutes) before positive staining appears, on the one hand, it may indicate that your antibody concentration is too low or the incubation time is too short The best primary antibody is 4 degrees overnight); on the other hand, the blocking time is too long. 12. Counterstaining: The purpose is to form a cell outline, so as to better locate the target protein, often counterstaining with hematoxylin (nuclear dye). Note that the hematoxylin counterstaining time depends on the room temperature, the old and new solutions, and the location of the target antigen. Generally, it takes a few seconds to a few minutes. The cytoplasmic protein can be longer and the nucleoprotein is shorter. But this can be remedied if the dyeing is not ideal. That is, the deeper staining can make the differentiation time a little longer; the lighter staining can be placed in hematoxylin and then stained. Hydrochloric acid alcohol is differentiated, ammonia water is blue. The role is different. After the film is counterstained, it is washed in running water, and then placed in hydrochloric acid alcohol for a few seconds (must be fast), then take out the running water and wash it, then put it back in the ammonia. 13. Mounting: For long-term storage, we generally mount with neutral gums to avoid air bubbles. The method is to apply a drop of mounting solution directly on the slide tissue, and then hold a corner of the coverslip in one hand, while the other Holding the opposite corner in one hand, the corner close to the proximal end of the mounting fluid is lowered until it comes into contact with the liquid; when the liquid contact surface is found to continue to disperse, you can slowly lower the other corner, so that generally no bubbles will be generated. Second, the important link in the immunofluorescence method 1. Preparation of frozen sections: It is recommended to use fresh tissue, otherwise the internal structure of the tissue cells will be destroyed, and the antigen will be easily dispersed. Choose clean and sharp blades, tissue must be frozen in moderation, etc., to prevent serious splitting and peeling. 2. Fixation of tissue slices: After the slices are air-dried, they are fixed with ice acetone or other fixative solution for 5-10min, especially for the white slices stored for a long time, they must be fixed in time and properly preserved. 3. Serum blocking: In order to prevent the binding of endogenous non-specific protein antigens, it is necessary to block with the serum (consistent with the source of the secondary antibody) before the primary antibody incubation, to weaken the background staining. Serum blocking time can be adjusted, generally 10-30min. 4. Primary antibody incubation conditions: the most important in the immunohistochemical reaction, including incubation time and antibody concentration. There are several kinds of primary antibody incubation temperature: 4 degrees, room temperature, 37 degrees, of which 4 degrees is the best; incubation time: this is related to temperature and antibody concentration, generally 37 degrees 1-2h, and 4 degrees overnight and taken out from the refrigerator After 37 degrees rewarming 45min. The specific conditions have to be explored. 5. Secondary antibody incubation conditions: the secondary antibody is usually at room temperature or 37 degrees for 30min-1h, the specific time needs to be explored, and the concentration generally has a working solution, if the concentrated solution is to explore the concentration, remember to avoid light reaction. However, in immunofluorescence, we generally set the concentration of the secondary antibody and the incubation time first, and then explore the concentration and incubation time of the primary antibody. Finally, the secondary antibody labeled with fluorescein may have a large amount of free fluorescein after the storage time is extended. It is necessary to pay attention to the small packaging and appropriate centrifugation when preparing. 6. Counterstaining: The purpose is to form a cell outline, so as to better locate the target protein. DAPI counterstain is commonly used. 7. Mounting: For long-term storage, we generally mount with buffered glycerin, etc. In addition, there is a special anti-fluorescence extraction and mounting solution. Avoid generating bubbles by dropping a drop of mounting solution directly on the slide tissue, and then holding a corner of the cover slip in one hand, and the opposite corner in the other hand, the corner close to the proximal end of the mounting solution is first lowered until When it comes into contact with the liquid; when it is found that the liquid contact surface is constantly dispersing, you can slowly lower the other corner, so that generally no bubbles will be generated. 8. Slice cleaning: In order to prevent the non-specific staining caused by the residual of primary antibody and secondary antibody, it is particularly important to strengthen the cleaning (extension time and increase the number of times). I generally clean before primary antibody incubation for 3min * 3 times , And the cleaning after the primary antibody incubation is 5 times * 5min. Note (1) Rinse separately to prevent contamination caused by cross-reaction. (2) Rinse gently to prevent the slice from falling off. I like to use the dip method; (3) The rinsing time should be enough to thoroughly remove the combined substances. (4) Use and requirements of PH and ionic strength of PBS. I have a painful lesson in this regard. At that time, the antibody dilution I bought was slightly acidic, and the result was a yellow background (no specific staining was seen). It is recommended that the pH is 7.4-7.6 and the concentration is 0.01M. (Neutral and weak alkaline conditions (PH7-8) are conducive to the formation of immune complexes, while acidic conditions are conducive to decomposition; low ionic strength is conducive to the formation of immune complexes, and high ionic strength is conducive to decomposition) 9 2. Photographing: If conditions permit, it is better to take a photo immediately. If you ca n’t take a photo in time, you should seal the film and seal it with nail polish to avoid light and humidity. When using a fluorescent microscope, pay attention to strictly follow the instructions of the fluorescent microscope factory instructions, do not change the program at will; check in a dark room; prevent UV damage to the eyes, wear protective glasses when adjusting the light source; the inspection time is 1 ~ 2h is appropriate, over 90min, the luminous intensity of the ultra-high pressure mercury lamp gradually decreases, and the fluorescence weakens; after the specimen is exposed to ultraviolet light for 3 ~ 5min, the fluorescence also significantly weakens or fades; the excitation light will decay and quench the fluorescence for a long time Phenomenon; therefore, it should not exceed 2 ~ 3h at most; the life of fluorescent microscope light source is limited, and specimens should be inspected intensively to save time and protect the light source. When it is hot, the fan should be added to cool down, and the new lamp should be recorded from the beginning. When the lamp is extinguished and you want to use it again, you can only ignite it after the lamp has cooled down sufficiently. Avoid lighting the light source several times a day. Turn off the mercury lamp at least 15-30 minutes after turning on; observe immediately after the specimen is stained, and the fluorescence will gradually weaken over time. If the specimen is stored in a polyethylene plastic bag at 4 ° C, it can delay the fluorescence weakening time and prevent the mounting agent from evaporating. The slides and other carriers used must have a uniform thickness and no obvious autofluorescence. If you use an oil lens, also It is necessary to ensure that the mirror oil is non-fluorescent mirror oil; the power supply is best equipped with a voltage stabilizer, otherwise the unstable voltage will not only reduce the life of the mercury lamp, but also affect the effect of the mirror inspection. Source: Bio Show Forum
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