European and American countries have successively carried out quality control of immunohistochemistry, and established a relatively complete set of quality control methods and procedures. The Chinese Pathologist's Committee (CCP) Immunohistochemistry Research Center draws on advanced foreign experience and combines with China's current actual situation to start exploring a method suitable for China's immunohistochemical quality control. At the same time, it discovers and promotes standardized staining procedures to improve And improve the reliability of the immunohistochemistry experiment, make the immunohistochemistry technology more standardized, and the results of the immunohistochemistry more reliable and repeatable. Although immunohistochemistry has been routinely carried out in many laboratories, it is an experimental method determined by multiple steps and factors. Due to the repair methods, staining methods, reagent manufacturers, antibody clone numbers, and detection systems adopted by each laboratory If there is a difference, the staining results will be quite different. A considerable number of laboratories have not paid enough attention to the standardization of immunohistochemistry, which has led to poor staining results misleading the pathological diagnosis, so the reliability of the immunohistochemical staining results Received great attention. There are many factors in the actual work that affect the results of immunohistochemistry, including whether the technical staff is proficient in the operation procedures of the entire experiment, the specificity of the antibody, the sensitivity of the method, the fixation of the specimen, the processing of the tissue, whether the antigen is repaired, Factors such as the PH value of the repair fluid. In order for each laboratory to continuously and reliably complete reliable experimental staining results, all processes in immunohistochemical staining technology should be standardized. 1. The factors of successful immunohistochemistry The pathologist must have considerable knowledge of immunohistochemistry diagnosis and experience in immunohistochemistry, and must clearly understand that immunohistochemistry is an experimental and highly technical technique. The expression of various antibodies in tissues and various factors that affect the results of immunohistochemistry can only improve the level of doctors' immunohistochemical diagnosis to avoid wrong judgment and ensure the accuracy of immunohistochemical diagnosis. Pathological technicians are the key factors for the success of immunohistochemistry experiments. Operators should have rich theoretical knowledge of immunohistochemistry and skilled immunohistochemistry staining techniques, and be very clear about the application principle of each step to make the experimental results more reliable. . Reliable experimental reagents and experimental methods are a must for every laboratory. Due to the variety of experimental methods and the variety of antibodies, each laboratory should ensure high-quality, high-efficiency reagents and explore the best experimental conditions. High-quality immunohistochemistry depends on effective antigen repair, sensitive detection systems, appropriate experimental quality control controls, and skilled and responsible technicians. Second, the immunohistochemical staining pre-processing technical operation specifications 1. Material selection: The ideal material thickness is 0.2cm ~ 0.3cm. Most laboratories believe that the main reason for tissue decoupling during the heating antigen repair process is due to excessive material thickness or improper dehydration and wax treatment. Good material collection, fixation, and dehydration processes are prerequisites for immunohistochemical quality control. If H & E sections fail to produce satisfactory staining results, then immunohistochemical staining will not be possible, and the quality of staining cannot be guaranteed at all. 2. Fixation: In many tissue fixatives (such as formaldehyde, ethanol, glutaraldehyde, paraformaldehyde, etc.), different experimental methods have their own advantages. However, formalin is the best, economical, and versatile in terms of maintaining the integrity of tissue and cell structure, antigen measurability, tissue permeability, and reagent prices. The fixatives containing acid or mercury are not ideal for antigen storage. Tissue fixation generally does not exceed 30 minutes after separation, and the fixation time is 8-24 hours under normal temperature. At the same time, we must also pay attention to avoid the loss of tissue antigen caused by over-fixation of formalin. Some studies have found that fresh tissue after formalin fixation for one week has a serious loss of tissue antigen, and the antigen can hardly be detected, because tissue fixation will cause protein or protein The formation of cross-links between molecules leads to the coverage of antigen sites, which can be corrected by antigen repair methods. If antigen repair is not used before adding antibodies, immunohistochemical staining often fails to achieve the desired results or is unsuccessful. The tissue can not be placed in 70% ethanol for a long time, the tissue shape after alcohol fixation is not as good as the formalin fixed tissue. The decalcification solution is more damaging to the antigen, so it is best to fix it in formalin solution for 48 hours before the tissue is decalcified. If the tissue is not fixed, the acid will destroy the antigen. The concentration of acid in the decalcification solution and the decalcification time Must be controlled to the lowest limit. 3. Immersion wax: We believe that the temperature of the immersion wax should be controlled at about 58 ~ 60 ℃, paraffin wax used for immersion wax should be replaced frequently to reduce the content of xylene in the paraffin wax. Xylene at high temperatures tends to make the tissue brittle, the cells shrink, and it is easier to drop pieces. 4. Section thickness: The section thickness used for immunohistochemical staining is 3-4 microns. In order to prevent stripping during the dyeing process, it is necessary to use siliconized glass slide mounts. 5. Preparation of siliconized slides: the slides are washed by acid washing and then baked; 2% APES immersed in acetone or anhydrous alcohol for 1-2 minutes; acetone or anhydrous alcohol for 1-2 minutes; distilled water for 1-2 minutes; Bake dry and set aside. 6. Grilled slices: sliced ​​slices are baked in the oven at 60 ℃ ～ 65 ℃ for about 30-60 minutes. Some laboratories use baked slices overnight. It has been reported that when the temperature of the roasted slices exceeds 60 ° C, the slices of the roasted slices for more than 18 hours have a slightly weaker immunohistochemical staining intensity than the slices baked for 4 hours. Too high temperature or too long time will affect the activity of the antigen, because it can accelerate the oxidation of the antigen in the tissue section under high temperature and dry conditions. 7. Section preservation: Some laboratory researchers are accustomed to continuously slicing tissue wax blocks for long-term storage at room temperature. After slicing tissues for long-term storage at room temperature, antigens can be lost. After discovering the wax block slices in the experiment, Maixin Lab will store them at room temperature for more than three months, and the results of immunohistochemical staining will be weakened or negative. A control experiment was conducted on the preservation time of new and old sections. Eleven different tissue wax blocks were selected, each with 10 consecutive sections, a total of 110 sections, placed in normal temperature air for one month, three months, half a year, one year and the new section. Paired control experiments. The experimental results showed that the tissue wax block was stored at room temperature for three months, and the sensitivity of the antigen to most antibodies decreased by about half, and some of the sections were lost more. Most of the antibodies stored in the slices for half a year cannot show positive labeling results. Only a few slices stored for more than one year can have weak positive expression, especially the loss of nuclear expressed antigen is more prominent. There are similar data reports abroad. The reason may be related to the oxidation in the air, so in actual work, wax seal slices can be used to avoid antigen loss for long-term storage, and can also be stored in the refrigerator at 4 ℃. Especially for slices used in scientific research experiments, pay attention to the slices. save. 8. Dewaxing: Immunohistochemical dewaxing steps are the same as conventional H & E dewaxing steps. In the immunohistochemical laboratory, dewaxing needs to be separated from H & E conventional dewaxing to ensure complete dewaxing, otherwise abnormal staining results may be caused. 3. Antigen retrieval in immunohistochemistry experiments Enzymatic digestion: such as trypsin, pepsin, proteolytic enzyme 2. Antigen heat repair: high-pressure method, microwave method and boiling method are well-known. The most critical part of immunohistochemical staining is antigen repair, and the repair of most commonly used antibodies is completed by heating. Hot antigen repair is better than enzyme digestion , More effective, the dyeing results are more consistent, and the operation is single. 3. Repair time: To obtain the strongest staining results, but also to maintain the integrity of the morphology. The problem with many antigen repairs is that when the tissue fixation time is too short, strong antigen repair can cause morphological damage, dissection, and complete tissue digestion. The solution can use a shorter time for heat repair or reduce the digestion time of the enzyme. 4. Antigen repair buffer: citrate buffer (pH6.0), Tris (pH7-8), EDTA (pH8.0 ～ 9.0), EGTA (pH9.0), etc., citrate buffer (pH6.0) ) The advantage is that the staining background is clear and suitable for most antibodies. The two repair solutions of Tris and EDTA have a strong repair effect on some antigens, but the staining background is also deepened. If used improperly, it is easy to cause false positive results. At present, there is no antigen repair solution that can be suitable for all antibodies. Citric acid buffer (pH6.0) can be used as an antigen repair buffer for routine immunohistochemistry, but it cannot be excluded that some antibodies are suitable for EDTA and EGTA buffers. For the repair solution, the antibody whose antigen is more difficult to express usually chooses a repair solution with a high pH value. For example: ER, PR, Bcl-2, Bcl-6, TdT, Ki-67, Cyclin D1, etc. 5. Precautions for antigen heat repair: Do not let the slice dry in any step. It needs to be cooled for 15-30 minutes after heating. Adequate antigen repair is the only important factor that affects staining results. Heating temperature and time can lead to insufficient or excessive repair. 6. The effect of antigen hot repair on endogenous biotin in tissues: While antigen hot repair enhances the expression of antigenic determinants, it also enhances the response of endogenous biotin in tissues. Using the Avidin-biotin detection system, the endogenous biotin in the tissue is prone to artificial artifacts, and the strong endogenous biotin can be observed in the negative pair photos with the same heating antigen treatment conditions. In response, there was no similar situation in the sections that had not been subjected to heat antigen treatment. Uniform, fine-grained, light brown positive in the cytoplasm, sometimes very strong expression, very similar to the expression of true positive results, in the past immunohistochemical staining endogenous biotin effect caused a lot of diagnosis Errors are often ignored. It is worth reminding that the concept must be very clear. The photo of the negative pair must be exactly the same as the antigen repair processing conditions of the primary antibody in order to accurately find the location of the endogenous biotin. 7. Endogenous biotin blocking method: generally, after antigen repair, biotin block treatment is performed with avidin before adding antibodies, and non-biotin detection systems such as EliVision, EnVision, SuperVesion, etc. can also be used. Fourth, the immunohistochemical staining experimental operating specifications 1. Dewaxing: xylene-→ alcohol-→ distilled water 2. Elimination of endogenous peroxidase: using peroxidase detection system, endogenous peroxidase must be blocked. Without treatment, the red blood cells and granulocytes in the tissue will interfere with the judgment of the staining results. Common use of 0.3% H2O2 for 10 minutes has no effect on staining results and antigen preservation, and the blocking effect is more significant. 3. Serum blocking: blocking with normal serum of the secondary antibody before adding the primary antibody can reduce non-specific binding. The two-step detection system currently used can eliminate this step. 4. Antibody use: There are a large number of ready-to-use antibodies for laboratory use. The manufacturer has conducted many experimental tests and can operate according to the conditions provided by the manufacturer. Concentrated antibodies are generally prediluted according to the recommended dilution provided by the manufacturer. Select the best dilution titer before conducting batch experiments. The deep staining background is mostly caused by high antibody concentration. The antibody incubation temperature is generally based on the normal temperature of 25 ℃ or 37 ℃ for 30 minutes, or overnight at 4 ℃ refrigerator. 5. Rinsing: The commonly used rinsing solution is PBS or TBS buffer solution. The rinsing step should be strictly implemented to prevent background coloring caused by unclean rinsing. Tween20 can be added to the buffer solution to enhance the rinsing effect. 6. Detection system: The universal detection system includes biotin-labeled ABC, SP, LSAB, etc. This type of detection system is more economical, and many hospitals are still using it. The non-biotin detection system is more expensive, because it does not need to be combined with biotin, it can avoid the interference of endogenous biotin. Its characteristics: sensitive, time-saving, convenient, and low background. 7. Color rendering system: Since the detection system uses horseradish peroxidase, DAB (brown) and AEC (red) are selected as substrates for the enzyme, and if the alkaline phosphatase system is used, BCIP / NBT (blue-violet) is selected The first choice for routine immunohistochemical color development is still DAB, with clear positioning and easy storage. AEC cannot tolerate alcohol and has low sensitivity. Although the BCIP / NBT positive is bright, the positive positioning is not accurate. 8. Counterstaining: The procedure of interlining dyeing is very simple, but the quality of interlining dyeing has a great influence on the quality of the final dyeing result. Some select Harris hematoxylin, and some use Mayer's hematoxylin, which has the best control effect with DAB staining and is easy to use in most laboratories. There are also laboratories that use methyl green as a lining dye, but they cannot fade with organic solvents. 5. Observation of staining results Positive results should be located in the corresponding parts of the cell, and positive results of antigens expressed on the cell membrane should be located on the cell membrane. Positive reactions in other parts are non-specific staining. Improper antigen repair can lead to changes in the localization of antigen in tissue cells. According to the different antigens detected, the antigens are located in: (1) cell membrane: such as LCA and CD3, CD20, etc .; (2) cytoplasm: such as Cytokeratin, GFAP, S100, CgA, AFP, etc .; (3) cell nucleus: such as Ki- 67. ER, PR, TTF-1, HPV-Ag, Cyclin D1, TDT, etc. 2. The periphery of the tissue, bubbles, knife marks, wrinkles and other parts often show non-specific positive expression. 3. Negative staining results do not always mean that the antigen is not expressed. Consider whether it is related to the destruction of the antigen in the tissue. 4. The deep background of the tissue section is related to the following factors: (1) The paraffin section is not cleanly dewaxed; (2) The concentration of the first antibody is too high or the incubation time is too long, the temperature is too high; (3) The concentration of the developer DAB is too high or H2O2 Too much; (4) The antibody is not pure; (5) The section of the antibody is not cleaned after incubation. 6. The immunohistochemical experiment control is to confirm whether the titers of the antibody and the test kit are reliable and whether the staining operation is correct. Generally, an experimental control is required to avoid false negatives and false positives due to reagent failure or improper operation to ensure the reliability of the staining results. Sex. 1. Positive control: Use tissue sections with known staining above moderate positive staining. The positive sections should be positive. In addition, the internal control in the tissue is also a good positive control. 2. Negative control: Use tissue sections that are known to be negative for staining, and the result should be negative. Vimentin staining was added as a control in each experiment, the purpose was to observe the sensitivity of the tissue to the expected antigen expression after formalin fixation, and to analyze the degree of antigen damage after formalin fixation. Vimentin is expressed in almost any tissue, and is especially suitable for immunohistochemical staining in biopsy tissues. There are a large number of vascular Vimentin positive expression in the interstitium connected to the epithelial tissue. If the Vimentin staining is weak or some areas become weak in the interstitium of the same slice, it indicates that improper fixation caused the destruction of antigen. Therefore, Vimentin staining should be routinely added in each batch of experiments to guide the observation of antigen expression sensitivity after formalin fixation. Seven, antibody storage and dilution Generally speaking, the antibody should be stored in a refrigerator at 4 ℃, the first antibody can be divided into small packages and stored at -20 ℃, stored at 4 ℃ when used, should not be repeatedly stored at 4 ℃ and -20 Between ℃, repeated freezing and thawing will make the antibody titer drop. The detection system is generally not suitable for storage at -20 ℃, because repeated freezing and thawing makes the enzyme bound to the antibody easy to decompose, resulting in reduced detection sensitivity. The concentrated antibody should be diluted according to the instructions or the best working concentration found by yourself before staining. The antibody can be diluted to 1:10, and then diluted into working solution before staining. Concentrate antibodies are stored for a longer period of time, whereas diluted antibodies are stored for a shorter period of time. If ready-to-use antibodies are used, after a certain period of time, pay attention to whether their titers are reduced to avoid false negative staining. Nestin and PDGFR can be used as diagnostic indicators for gastrointestinal stromal tumors, especially for the diagnosis of CD117 negative gastrointestinal stromal tumors, and have been reported in foreign literature. These two antibodies can be used in conventional fixed paraffin sections. Experiments have shown that Nestin uses PH8.0 EDTA high-pressure repair, and PDGFR uses PH6.0 citric acid microwave repair. Source: Lilac Garden Forum
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