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Immunohistochemical specification
Immunohistochemical specification
European and American countries have carried out immunohistochemical quality control work, and established a relatively complete set of quality control methods and procedures. The Chinese Society of Pathologists (CCP) Immunohistochemistry Research Center draws on the advanced experience of foreign countries and combines the current situation in China to explore a method suitable for quality control of immunohistochemistry in China. At the same time, it discovers and promotes standardized dyeing procedures and improves And to improve the reliability of immunohistochemistry experiments, the immunohistochemistry technology is more standardized, and the results of immunohistochemistry are more reliable and reproducible. Although immunohistochemistry has been routinely carried out in many laboratories, it is a multi-step, multi-factorial assay method, due to the repair methods, staining methods, reagent manufacturers, antibody clone numbers, and detection systems used in each laboratory. If the results are different, the staining results will be greatly different. A considerable part of the laboratory does not pay enough attention to the understanding of immunohistochemistry, which leads to misleading results of pathological diagnosis, so the reliability of immunohistochemical staining results. Received great attention.
In practice, there are many factors that influence the results of immunohistochemistry, including whether the technician is proficient in the entire experimental procedure, antibody specificity, method sensitivity, specimen fixation, tissue processing, antigen retrieval, The pH of the repair fluid and other factors. In order for each laboratory to consistently and reliably perform reliable experimental staining results, all processes in immunohistochemical staining techniques should be standardized.
First, the elements of successful immunohistochemistry
Pathologists should have considerable experience in immunohistochemistry and immunohistochemistry. It is necessary to understand that immunohistochemistry is an experimental and highly technical technique. It is necessary to understand the expression of multiple antibodies in tissues. And various factors affecting the results of immunohistochemistry, only to improve the doctor's immunohistochemical diagnosis level can avoid false judgments, in order to ensure the accuracy of immunohistochemistry diagnosis. Pathological technicians are the key factors for the success of immunohistochemistry experiments. Operators should have rich theoretical knowledge of immunohistochemistry and skilled immunohistochemical staining techniques. The application principle of each step is very clear, and the experimental results are more reliable. . Reliable laboratory reagents and experimental methods are essential for each laboratory. Due to the variety of experimental methods and the wide variety of antibodies, each laboratory should ensure high quality and high cost reagents and explore the best experimental conditions. Good quality immunohistochemistry depends on effective antigen retrieval, sensitive detection systems, appropriate experimental quality control controls, and skilled and responsible technicians.
Second, immunohistochemical staining pretreatment technology operation specification
1. Material : The ideal material thickness is 0.2cm ~ 0.3cm. Most laboratories believe that the main reason for the tissue to cause stripping during the heating antigen repair process is due to excessive material extraction or improper treatment of dehydrated wax. Good process of obtaining, fixing and dehydrating is the premise of immunohistochemical quality control. If H&E sections can not produce satisfactory staining results, immunohistochemical staining will not be discussed, and the quality of dyeing cannot be guaranteed.
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, both economical and versatile in maintaining tissue cell integrity, antigen measurability, tissue permeability, and the price of reagents. The acid or mercury-containing fixative is not ideal for antigen preservation. The tissue should not be fixed after being in vitro for more than 30 minutes, and the fixed time is 8-24 hours under normal temperature conditions. At the same time, we should also pay attention to avoid the loss of tissue antigen caused by formalin over-fixation. Some studies have found that fresh tissue is severely lost after one week of formalin fixation, and the antigen can hardly be detected, because the tissue will cause protein or protein after fixation. Crosslinking between molecules leads to antigenic site occlusion, which can be corrected by antigen retrieval methods. If antigen retrieval is not used before antibody addition, immunohistochemical staining often fails to achieve ideal results or is unsuccessful. Tissues should not be placed in 70% ethanol for long periods of time. The tissue morphology after alcohol fixation is not as good as that of formalin-fixed tissue. Decalcification solution is more serious for antigen destruction. Therefore, it is best to fix it in formalin for 48 hours before decalcification. If the tissue is not fixed, the acid will destroy the antigen, the acid concentration in the decalcification solution and the decalcification time. All must be controlled to the lowest possible limit.
3. Dipping wax : We believe that the temperature of the dipping wax should be controlled at about 58-60 °C. The paraffin wax used for dipping wax should be replaced frequently to reduce the content of xylene in paraffin. Xylene at high temperatures tends to make the tissue brittle, the cells shrink, and it is easier to fall off.
4. Slice thickness : The thickness of the section used for immunohistochemical staining was 3-4 microns. To prevent stripping during the dyeing process, a siliconized slide is required.
5. Preparation of silicified slide : The slide is rinsed by pickling and dried; 2% APES acetone or absolute alcohol soak for 1~2min; acetone or anhydrous alcohol for 1~2 minutes; distilled water for 1~2min; Bake dry and spare.
6. Baked slices : Slices are baked in an oven at 60 ° C ~ 65 ° C for about 30-60 minutes. Some laboratories use baked slices overnight. It has been reported that when the temperature of the baked sheet exceeds 60 ° C, the slices of the baked sheet are more than 18 hours, and the immunohistochemical staining intensity is slightly weaker than that of the grilled for 4 hours. Excessive temperature or excessive time can affect the activity of the antigen, because the oxidation of the antigen in the tissue section can be accelerated under high temperature drying conditions.
7. Slice preservation : Some laboratory researchers are accustomed to long-term preservation of tissue wax blocks after continuous sectioning at room temperature, and the tissue after slicing can be preserved for long-term preservation of antigen at room temperature. In the experimental laboratory, the wax block was found to be stored at room temperature for more than three months, and the results of immunohistochemical staining were weakened or negative. The preservation time comparison experiments of new and old sections were carried out. Ten pieces of 11 different tissue wax blocks were selected and serially sliced ​​into 10 pieces, 110 pieces in total, and placed in normal air for one month, three months, half a year, one year and new sections. Paired control experiments. The experimental results show that the tissue wax block is stored at room temperature for three months, the sensitivity of the antigen to most antibodies is reduced by half, and some slices are lost more. The antibody that was preserved for half a year in the slice could not be positively labeled, and only a few slices stored for more than one year could have weak positive expression, especially the antigenic loss of nuclear expression was more prominent. There are similar reports in foreign countries. The reason may be related to the oxidation in the air, so in the actual work, wax seals can be used to avoid antigen loss for long-term preservation, or can be stored in a refrigerator at 4 °C, especially for the sections used in scientific research experiments. save.
8. Dewaxing : The immunohistochemical dewaxing step is the same as the conventional H&E dewaxing step. Dewaxing in the immunohistochemical laboratory needs to be separated from H&E conventional dewaxing to ensure complete dewaxing, otherwise it may lead to abnormal staining results.
3. Antigen retrieval in immunohistochemistry experiments
1. Enzymatic digestion : such as trypsin, pepsin, proteolytic enzymes
2. Antigen heat repair : high pressure method, microwave method, boiling method
It is well known that the most critical factor in immunohistochemical staining is antigen retrieval, and the repair of most commonly used antibodies is done by heating. Thermal antigen retrieval is superior to enzymatic digestion, more effective, and the staining results are more consistent and uniform.
3. Repair time : both the strongest staining results and the integrity of the tissue morphology. The problem with many antigen retrieval is that when the tissue fixation time is too short, strong antigen retrieval can cause morphological destruction, stripping and complete tissue digestion. The solution can be used for a short time of heat repair or to reduce enzyme digestion time.
4. Antigen repair buffer : citrate buffer (pH6.0), Tris (pH7-8), EDTA (pH 8.0 ~ 9.0), EGTA (pH 9.0), etc., citrate buffer (pH6.0 The advantage is that the staining background is clear, suitable for most antibodies, Tris and EDTA two kinds of repair liquids have a stronger effect on partial antigen repair, but the staining background is deepened at the same time, such as the use of improper detection of false positive results. At present, there is no antigen-repairing solution suitable for all antibodies. Citric acid buffer (pH 6.0) can be used as an antigen retrieval buffer for immunohistochemistry, but it cannot be excluded. Some antibodies are suitable for EDTA and EGTA buffering. Repair fluids, generally more difficult to express antibodies, more high-pH repair solution. For example: ER, PR, Bcl-2, Bcl-6, TdT, Ki-67, Cyclin D1, and the like.
5. Precautions for antigen heat repair : Do not let the slices dry out in any step. After heating, it needs to be cooled for 15-30 minutes. Adequate antigen retrieval is the only important factor affecting the results of staining, and the temperature and time of heating can lead to under-repair or over-repair.
6. Effect of antigen heat repair on endogenous biotin in tissues : Antigen heat repair enhances the expression of endogenous biotin in tissues while enhancing the expression of antigenic determinants. Using the Avidin-biotin detection system, endogenous biotin in tissues is prone to human artifacts, and strong endogenous biotin can be observed in negative photographs with the same heating antigen treatment conditions. The reaction did not show a similar situation in the sections without thermal antigen treatment. In the cytoplasm, it is uniform in the form of fine-grained light brown, sometimes very strong, and is very similar to the expression of true positive. The influence of endogenous biotin in the previous immunohistochemical staining has caused many diagnoses. Errors are often ignored. It is worth reminding that the concept must be very clear, the negative photo must be exactly the same as the antigen retrieval process of the primary antibody, in order to accurately find the site of endogenous biotin.
7. Endogenous biotin blocking method : Generally, after antigen retrieval, avidin is used for biotin blocking treatment before adding antibody, and non-biotin detection systems such as EliVision, EnVision, SuperVesion, etc. can also be used.
Fourth, immunohistochemical staining experiment operation specification
1. Dewaxing : xylene - → alcohol - → distilled water
3. Serum occlusion : Closure with normal sera from a secondary antibody prior to the addition of the primary antibody reduces non-specific binding. The two-step detection system currently in use can be eliminated from 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 be operated according to the conditions provided by the manufacturer. The concentrated antibody is generally subjected to a double dilution pre-experiment according to the recommended dilution provided by the manufacturer. Batch experiments were performed after selecting the optimal dilution titer. The depth of the staining background is mostly due to the high antibody concentration. The incubation temperature of the antibody is generally based on a normal temperature of 25 ° C or 37 ° C for 30 minutes, or a refrigerator at 4 ° C overnight.
5. Flushing : The commonly used rinsing solution is PBS or TBS buffer. Strictly perform the rinsing step to prevent background coloration caused by improper rinsing. Adding Tween20 to the buffer can enhance the rinsing effect.
6. Detection system : The universal detection system has biotin-labeled ABC, SP, LSAB, etc. This kind of detection system is more economical, and there are still many hospitals in use. Non-biotin detection systems are more expensive. Since there is no need to combine biotin, the interference of endogenous biotin can be avoided. Its characteristics are: sensitive, time-saving, convenient, and low background.
7. Color development system : Since the detection system uses horseradish peroxidase, DAB (brown) and AEC (red) are selected as substrates for the enzyme, and if alkaline phosphatase system is used, BCIP/NBT (blue-violet) is selected. The preferred immunohistochemical color development is still DAB, which is clearly positioned and easy to store. AEC can not tolerate alcohol and has low sensitivity. Although BCIP/NBT positive is bright, the positive positioning is not accurate.
8. Counterstaining : The lining step is very simple, but the quality of the lining has a great influence on the quality of the final result of the dyeing. Some use Harris hematoxylin, some use Mayer's hematoxylin, and the best contrast with DAB staining, easy to use in most laboratories. There are also laboratories using methyl green lining dyeing, but not organic solvents, easy to fade.
V. Observation of dyeing results
1. The positive result should be located in the corresponding part of the cell, and the positive result expressed in the cell membrane should be located on the cell membrane, and the positive reaction in other parts is non-specific staining. Improper antigen retrieval results in changes in the localization of antigens in tissue cells. Depending on the antigen being detected, the antigens are located at:
(1) Cell membrane: such as LCA and CD3, CD20, etc.;
(2) Cytoplasm: such as Cytokeratin, GFAP, S100, CgA, AFP, etc.;
(3) Nuclei: such as Ki-67, ER, PR, TTF-1, HPV-Ag, Cyclin D1, TDT, and the like.
2. The periphery of the tissue, bubbles, knife marks, wrinkles and the like often exhibit non-specific positive expression.
3. Negative staining results are not all antigens not expressed, and should be considered whether it is related to the destruction of antigens in tissues.
4. The depth of the tissue section is related to the following factors:
(1) Dewaxing of paraffin sections is not clean;
(2) The concentration of the first antibody is too high or the incubation time is too long, and the temperature is too high;
(3) The concentration of DAB of the developer is too high or too much H2O2;
(4) The antibody is impure;
(5) The sections were not cleaned after the antibody was incubated.
Sixth, immunohistochemistry experiment control
In order to confirm whether the antibody and the test kit are reliable and the staining operation is correct, it is generally necessary to carry out an experimental control to avoid false negatives and false positives due to reagent failure or misoperation, and to ensure the reliability of the staining result.
1. Positive control : stained with tissue sections with more than moderate positive staining, positive sections should be positive, and the internal control in tissues is also a good positive control.
2. Negative control : stained with tissue sections that are known to be negative for staining, the results should be negative. Vimentin staining was added as a control in each experiment to observe the sensitivity of the expected antigen expression of the tissue after formalin fixation, and the extent of antigen damage after formalin fixation was analyzed. Vimentin is expressed in almost any tissue, especially for immunohistochemical staining in biopsy tissues. There is a large number of vascular Vimentin positively expressed in the interstitial connected with the epithelial tissue. If Vimentin staining is weak or part of the region is weakened in the interstitial of the same section, it indicates that improper fixation leads to antigen destruction. Therefore, Vimentin staining should be routinely added in each batch to guide the observation of antigen expression sensitivity after formalin fixation.
Seven, antibody preservation and dilution
In general, antibodies should be stored in a refrigerator at 4 ° C, the first antibody can be stored in small packages at -20 ° C, stored at 4 ° C when used, should not be stored repeatedly between 4 ° C and -20 ° C, repeated freezing and thawing Will cause antibody titers to fall. The detection system is generally not suitable for storage at -20 ° C, because repeated freeze-thaw cycles allow the enzyme bound to the antibody to be easily decomposed, resulting in reduced sensitivity of the assay. Concentrated antibodies should be diluted prior to staining according to the instructions or at the optimum working concentration. The antibody can be diluted to 1:10 and diluted to a working solution before staining. The concentrated antibody is stored for a longer period of time. On the contrary, the diluted antibody has a shorter shelf life. If a ready-to-use antibody is used, after a certain period of time, it should be noted that the titer is 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. It has been reported in foreign countries. These two antibodies can be used for routinely fixed paraffin sections. It has been proved that Nestin is repaired with PH8.0 EDTA high pressure, and PDGFR has better microwave repair with PH6.0 citric acid.