Common problems and solutions of immunohistochemistry 1. After the primary antibody is taken out from 4 degrees, why do we need to re-warm it at 37 degrees? (1) On the one hand, prevent the slices from being put into PBS easily from 4 degrees to take off the slice; (2) On the other hand, make antigen-antibody binding more stable. Generally not needed, but may be useful for weakly expressed antigens. The molecular motion is different at 4 degrees and 37 degrees. The former has a lower probability of collision and faster movement than the latter. The latter combines faster, but the sensitivity is also improved and easy Causes non-specific staining. (3) Actually, I agree with the latter statement, because I tried to take the liver or testicle slices from 4 degrees overnight and washed them directly with PBS. 2. The background is too dark after staining, how to distinguish between specific and non-specific staining? Full coloring means that the entire slice is stained with color, and the intensity of the coloring can be dark or light. In short, it is not clear which tissues are positive and those tissues are negative. The reasons for this phenomenon are: (1) The antibody concentration is too high: the primary antibody concentration is too high is one of the common reasons. The solution is to test the working concentration of each new antibody before using it, so that each antibody can be personalized and find the ideal working concentration for its own laboratory, even if it is a ready-to-use antibody, it ca n’t be simple According to the instructions for dyeing. (2) The antibody incubation time is too long or the temperature is high: The solution is to strictly implement the operating procedures. It is best to wear a timepiece or clock with you to remind you in time to avoid prolonging the time due to forgetting. The popular two-step method (Polymer) is highly sensitive, requiring the primary antibody incubation time to be not 1 hour, but 30 minutes. Therefore, it should be adjusted according to the staining result. (3) DAB deterioration and color development time is too long: DAB is best used now, if there is sediment, it should be filtered before use. The prepared DAB should not be stored for too long, because in the absence of enzymes, hydrogen peroxide will also release oxygen atoms and react with DAB to reduce the effectiveness of DAB. Unused DAB is stored in the refrigerator for a few days. It is not advisable to use this seemingly economical method. The color development of DAB is best monitored under a microscope, and the reaction is terminated immediately when the desired degree of staining is reached. However, when there are too many stained sheets or when using a staining machine, this seems unrealistic, but at least some new or less used antibodies should be monitored for color development to avoid excessive color development time. (4) Dried tissues: Failure to replenish the fluid in a timely manner after the repair fluid overflows, too many stained slices, too slow movements, forgetting to drip, and dripping of the drip are all reasons that cause the tissue to dry out. The solution is to operate carefully, using DAKO pen or PAP Pen to draw a circle around the tissue, which can effectively avoid the loss of liquid and increase the operation speed. (5) The immersion time of the slice in the buffer or repair solution is too long (more than 24 hours): the reason is not clear, but the phenomenon exists. Some laboratories like to dewax the slices to repair the day before, and add antibodies for immunohistochemical staining the next day. If the container with slices and repair solution is placed in a 4oC refrigerator overnight, there is no obvious effect on the results. At room temperature, especially on hot summer days, background coloring will appear, so it should not be stored for too long. (6) Polyclonal antibodies with degraded primary quality and poor quality: Pay attention to the expiration date of the antibody. The expired antibody is either not colored or the background is colored. When using a newly purchased antibody, it is best to set up a positive control and compare it with the used antibody. 3. What are the reasons for the release? (1) The quality of polylysine slides. I originally bought it, and it is said that the new brand is also good, but what it looks like. It is better to supplement the film made by the pathology teacher used in the second batch later. (2) The tissue is not cut well. The problems of the slicing machine are, for example, the thickness of the old machine is not uniform, or the slicing technique is not good. (3) It is not baked well, the time is short, and the temperature is not enough. (4) When the operation is too sharp, it is best not to shake or gently shake the film that is suspected of being removed, and slowly absorb water from the edge with toilet paper. (5) Repair problem: When the antigen is repaired, the high-pressure time is too long, or the technique is not good when put into the 100-degree repair solution, and it is thrown into it with a bang. In addition, EDTA repair is easier to take off than citric acid, but when you want to use EDTA, there is no way to do it, only from other issues. (6) Once you see the organized floating operation, you should be more cautious. Try to use PBS when using PBS, do not flush. Basically, I have paid attention to these aspects. As far as possible, I can improve it as much as possible. (7) The problem of tissues. I use a lot of cancer tissues. The more cancer tissues have necrosis, the easier it is to take off. 4. What are the best conditions for antigen repair in immunohistochemistry? (Especially microwave repair) Regarding antigen repair, the repair conditions I have tried include EDTA thermal repair, microwave repair using sodium citrate as a buffer, and autoclave repair. From my experimental results, microwave repair is not easy to drop the film, and EDTA hot repair and pressure cooker repair are very easy to drop the film. Because the film drop is mainly caused by the bubbles generated during the heating process, and the microwave repair water is heated to boiling, there will be less air bubbles on the film, and it will not cause off-chip due to the small bubbles on the string. When doing EDTA repair, you can put the slide as close as possible, or place some cotton or something around the glass slide, and then cover the cover slip, so that the repair can minimize the formation of small bubbles on the slide. The microwave repair conditions we used are: 2.94g trisodium citrate dissolved in 1000ml of water, adjust the PH value to 6.0, microwave repair for 10 minutes. You can try it, the time can be adjusted according to your needs. For citric acid repair, as long as the high-pressure repair time is controlled to 90 seconds after the valve is added to the air, the pressure cooker cover is opened under cold water. High-pressure repair does not drop the film like microwave repair, and the general high-pressure repair effect is often better; About EDTA For hot repair, the general manual says that the PH value is equal to 9. Actually, I tried it. If EDTA is used for single pass, it is easy to drop the film, but with Tris-EDTA, the film is generally not dropped. The concentration of Tris-base It is 0.05mmol / L, both high-pressure and microwave repair are possible, and the PH is also equal to 9. 5. After DAB color development, it is found that the coloring of the slices is uneven: if one piece is colored, one piece is not colored or the staining is light? The uneven coloring may have a lot to do with your experimental methods, and the possible reasons are: (1) The thickness of the cut piece itself may be different. Cut out a thick and a thin piece, which may cause different shades of color. (2) It may be that the substrate of your coloring solution is not shaken evenly after it is added to the film, resulting in inconsistent local substrate concentration. Therefore, after adding the coloring solution, it is best to shake the film back and forth a few times to make all of the film The substrate concentration in the area is consistent. (3) If your film is dyed deep in the middle and light near the edge, it may be that your wax circle painting is too small, and the area covered by the coloring liquid is not too large and has an edge effect. The outermost tissue piece is best 0.5cm away from the outer edge of the developing solution. Mat Series,Dough Mat,Silicone Dough Mat,Baking Mat Changshu Xinneng Silicone Products Co., Ltd. , https://www.xnsilicone.com
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