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When formalin is immobilized, many amino acid residues in the tissue form aldehyde bonds in or between molecules, causing many antigenic determinants to be blocked. At the same time, due to the polymerization of formaldehyde, the cross-linking of protein molecules to form a macromolecular network also leads to the masking of antigenic determinants, which makes a considerable part of the antigen unable to react well with the antibody. Therefore, antigen retrieval is also an essential factor affecting the results of immunohistochemical staining. Antigen retrieval (AR) technology, established in a series of biochemical studies by Fraenkel-conrat et al. [1], developed by Shi et al. [2] in 1991. Antigen retrieval refers to paraffin, frozen, fire-cotton gum, plastic sections immunohistochemistry (IHC) before trypsin, urea, surfactants, microwave buffers and metal salts, etc., to mask the antigenic determinants or denatured antigens Re-exposure or antigenicity results in a degree of recovery. Antigen retrieval can restore the antigenicity lost during the process of tableting and the like, and many antibodies that were originally thought to be unable to perform IHC on paraffin sections have obtained good staining results and become an effective remedy. The purpose of this paper is to outline the development, application, and standardization of AR-HIC.
First, AR technology
Heating AR technology
Microwave heating method. In the traditional standard method, dewaxing, dehydration and blocking of endogenous peroxygenase enzymes with 3% H2O2, paraffin sections were rinsed with distilled water and placed in a plastic Coplin cylinder. Add AR solution, such as distilled water, buffer, metal salt solution, urea, etc., cover and stand in a household microwave oven for 5 or 10 minutes (be careful to prevent slicing). Sometimes after heating for 5 minutes, the interval was 1 minute and the heating was continued for 5 minutes (the liquid height was measured after the first 5 minutes). After heating, the plastic Coplin cylinder was removed from the microwave oven and allowed to cool for 15 minutes. The sheet was washed twice with distilled water and then immersed in PBS for 5 minutes before IHC staining. In order to maintain consistent heating conditions, it is necessary to set the same position and the same number of Coplin cylinders near the center of the microwave oven, set different heating times according to different antibodies, and establish standard heating conditions as much as possible.
The microwave (MW) heating process is as follows: 200 ml of citric acid buffer (pH 6.0 ± 0.1) is placed in a special box, and a lid with a small hole is covered, and the microwave is heated to boiling; the tissue after dewaxing is hydrated. The slices were placed in a high-temperature plastic slice holder and placed in a boiling buffer. The authors provided a microwave oven with a medium power of 800 W [3] for immunohistochemistry and a domestic microwave oven with a defrosting file for microwave ovens (according to current research, recommendations). Use a medical microwave oven), continue to process for 10-20 minutes in the mid-range; remove the microwave plastic cylinder and cool to room temperature, remove the slide from the buffer, rinse the sheet twice with distilled water, then rinse twice with PBS (pH 7.2-7.4). 3 minutes each time.
Although a few authors [4] believe that the step of cooling after high temperature is omitted. In our opinion, there are several reasons why 15 minutes of cooling are necessary: 1. If this step is omitted, for some antibodies, the intensity of AR-IHC staining will be reduced. 2, suddenly from high temperature to low temperature can cause tissue pieces to fall off. 3. May cause morphological changes.
Simple heating method. Place the slices in a container containing citric acid buffer and heat to boiling in an electric furnace (600W) for 10 minutes. Shi et al. [2] compared IHC staining intensity with a simple heating method and a microwave heating method, showing that both methods result in the same staining intensity.
High-pressure heating method. Shin et al. [5] developed an aqueous autoclave boiling method to enhance the tau protein immunoreactivity of formalin-fixed brain tissue. Place the slice together with the citrate buffer in the pressure cooker, heat until a jet is generated, and continue for 2 minutes. The pressure cooker leaves the heat source. The control of the length of the heating is very important. The total time from the tissue section into the buffer to the pressure cooker to the pressure cooker leaves the heat source at 5- 8 minutes, too long may make the staining background deeper. Most of the antibodies provided by the reagent company are recommended to use the autoclave boiling method. The practice in recent years has shown that this method can avoid false positives and negatives, and the IHC dyeing effect is better.
Non-heating AR technology
Non-heated AR techniques include methods such as enzymatic digestion, acid hydrolysis, and the like. At present, mainly enzyme digestion, enzyme digestion is a chemical method to break the aldehyde bond and repair the antigen.
Trypsinization. Add 0.05g or 0.1g trypsin to 100ml of 0.05% or 0.1% pH 7.8 anhydrous calcium chloride solution to dissolve; or 0.125% liquid trypsin provided by Maixin. The sections were placed in a 37oC incubator in a wet box for 18-20 minutes.
Pepsin digestion method. Prepare 0.4% pepsin with 0.1 mol/L HCL;
Pronase digestion method. Pronase was dissolved in 0.5 mol/L Tris-HCL pH 7.4 at a concentration of 0.1% and a digestion time of 20 min.
The choice of antigen retrieval method is related to whether the tissue antigen can be fully exposed, which has a great influence on the immunohistochemical staining effect. Therefore, different repair methods should be adopted according to different antigen characteristics. For certain intracellular antigens (such as Fas, Bax, FVIII, etc.) and interstitial antigens (such as Laminin, CoIV, etc.), it is necessary to treat the sections by enzymatic digestion. Commonly used digestive enzymes are trypsin and pepsin. In general, trypsin digestion is weaker than pepsin, and is mainly used for digestion of intracellular antigens. Pepsin is mainly used for digestion of intercellular antigens. In the work, we must carefully refer to the instructions of the antibody instructions for the selection of digestive enzymes, so that the targeting is stronger and the labeling effect is more ideal.
In short, the high-pressure heating method and the microwave heating method are relatively stable, and the high-pressure heating method is superior to the microwave heating method and the simple heating method. The concentration of the solution has no effect on the repair effect, while the pH value has a greater influence. Buffers are more commonly used in citrate buffers and Tris-HCL. Previous studies have shown that high temperature repair time is short [6]. The 251st Hospital of the People's Liberation Army based on the clinical needs of pathological diagnosis, differential diagnosis and research, based on the research and application of antigen retrieval methods, the use of trypsin-urea combined digestion technology, hydrochloric acid hydrolysis exposure antigen technology and MW-surface activity Triton X-100 (MW-TX) repair antigen technology [7]. There are many antigen exposure or antigen retrieval methods, among which MW-decanoic acid buffer is used more and the effect is best. The method for repairing antigen by MW is to dewax the paraffin section, put it into the repairing liquid after washing with water, and use the output power of 250W for 2X5min, and cool it to room temperature and treat it as usual. The current AR process has been successfully applied to the BioTek automated immunohistochemical stainer.
Second, the AR should pay attention to the problem
Heating duration and strength are important factors, and the pH, concentration and chemical composition of AR fluid are also important factors. Negative pair photos must be used with low pH AR fluids to prevent misalignment [9]. Shi et al. [10] emphasized that the pH of the repair buffer is important for the repair of certain antigens. In the experiment, we also found that the optimal pH value of the antigen retrieval solution must be selected in order to obtain satisfactory results. In the conventional paraffin section, AR-IHC was used, and different concentrations of Alcl3 solution were used as the AR solution. It was found that 4% of Alcl3 achieved the best staining [11]. Among the repaired antigens, metal salts can affect the structure of the protein. Zheng Hui et al [12] used 0.01mmol / L PBS, 0.01mol / L citrate buffer, 0.1mol / L Tris-HCL buffer and 1mmol / L EDTA-Na2, repaired the antigen in the microwave, and found that its positive intensity gradually increased .
Due to the complexity of the mechanism of high temperature antigen retrieval, it is difficult to solve some existing problems by design control methods. Some antibodies have a negative reaction on frozen sections, but they can be well displayed after paraffin sections are repaired with antigen. For some antigens that should be expressed in the cytosol or expressed in the nucleus, most of them are expressed in the nucleus after repairing, for example, P185, Bcl-2, P-gp, Nm23, and some are expressed in the nucleus. The antigen is repaired in the cytoplasm, such as P53, PcNA, Ki-67, etc. [6]. Care must be taken when using this method, and the judgment of the results should be objectively analyzed. Hou Ning et al. found that telomerase reverse transcriptase (TRT) has different staining effects after antigen retrieval and no antigen retrieval, the latter The positive rate was significantly higher than the former [8]. Attention should also be paid to the following problems during operation:
1. Pay attention to natural cooling after heat treatment.
2. Prevent the slices from drying during heat treatment.
3. Appropriate antigen retrieval methods should be selected according to different antibodies.
4. The temperature and time of a batch of antigens must be consistent.
5, pay attention to the change of morphological structure (generally after the high temperature repair, the cytoplasm will be obviously destroyed, but the impact on the nucleus is greater, there will be nuclear rupture, hematoxylin staining, etc., and enzymatically hydrolyzed sections, cells Pulp destruction varies depending on digestion time, but nuclear damage is lighter).
6. If antigen retrieval cannot be achieved in the commonly used buffer, or the antigen localization changes after repair, some unusable buffers may be used, or some chelating agents such as EDTA may be used to improve the repair of certain antigens.
There are many enzymes used for IHC digestion. The type of enzyme selected, the concentration used, the pH value, the digestion time and the temperature are all different depending on the tissue fixation. The nature of the antigen to be detected and the type of tissue will vary. Experiment and find out. The principle of using enzyme digestion: trypsin and proteinase K are generally used for intracellular antigens such as Keratin, CEA, GFAP and the like. Pepsin is mainly used for the detection of interstitial antigens, such as fibronectin, Laminin, various types of collagen. Generally speaking, the digestion time is proportional to the length of tissue fixation. In the case of enzymatic digestion and heat recovery, the combination of antigen retrieval and enzymatic digestion is used, and sometimes satisfactory results are obtained. In general, proteinase K and microwave are combined, but it should be noted that any antigen that may be detected will have a false positive reaction in the nucleus regardless of its nature [6].
Third, the clinical application of AR.
In the early 1990s, with the development of AR technology, IHC applied a new field in understanding the human pathogenesis and pathophysiology at the molecular level on the human tibia embedded in the colloidal cotton. ]. Immunohistochemical staining of IC5 and ID15 monoclonal antibodies was assessed on conventionally processed paraffin sections using the AR method [14]. Charalambous. C et al. [15] strongly recommended the MW-AR-IHC method to detect CD30 in R-S cells. AR technology has been successfully applied to in situ hybridization [16]. Using heating AR-IHC technology, nestin was detected on formalin-fixed, paraffin-embedded sections with antitoxin #4350 [17]. Immunohistochemical localization of DCC protein in human epithelial, neurological, and neuroendocrine tissues using anti-complex techniques [18]. AR-IHC has been widely used in clinical research.
Fourth, the standardization and development of AR
The exact principle of antigen retrieval is not very clear. According to domestic and foreign literature reports, it is speculated that: 1. Dissolve and elute denatured proteins. 2. Hydrolysis. 3. The repairing effect of direct collision of ions or polar molecules in the microwave field [7]. Is antigenic repair possible? Theoretical research is not yet clear, but the experience of high temperature, high pressure and antigen retrieval treatment on conventional paraffin sections shows that the positive detection rate of antigen and antibody can be improved, and false positives can also occur.
The diagnosis of AR technology on IHC and its research in the basic field has been described in numerous literatures and more reviews. The purpose of the investigation in this field is to form new molecular morphology from the molecular diagnostics routinely, the possibility of detecting new pathways of nucleic acids and proteins in paraffin tissue. Some new approaches must establish principles of standardization and increase their repetitiveness. [19] . At present, the pathology department of most hospitals in China has not been standardized on IHC and AR. The standardization of AR and the standardization of IHC have been widely carried out abroad [20]. For example, in the establishment of new repair fluids, the heating conditions and liquid concentration, pH value, and chemical composition are predefined for different antibodies, thereby promoting the standardization of AR.
AR technology has been widely used in various fields of immunohistochemistry (IHC), such as in diagnostic pathology and IHC standardization. For antigens originally expressed on frozen sections, most of the antibodies after AR can be used on conventional formaldehyde-fixed paraffin sections, and the IHC retrospective study and clinical pathological IHC differential diagnosis, prognosis detection and efficacy of tumor patients after surgery Evaluation has a positive meaning.
references
1 Fraenkel-conrat H, Olcott HS .The raction of formaldehyde with proteins. V. Cross-linking between amino and primary amide or guanidy1 groops.J.AM.Chem.Soc.1948;70:2673-2684
2 Shi SR,Key ME, Kalra KL..Antigen retrieval in formalin-fixed,paraffin-embedded tissues:An enhancement method for immunohistochemical staining based on micromwave oven heating of tissue sections.J.Histochem.Cytochem. 1991;9:741- 748
3 Swanson PE. Microware antigen retrieval in citrate buffer.Lab.Med. 1994;25:520-522
4 Geradts J, Hu SX, Lincoin CE, et al. Aberrant RB gene expression in routinely processed, archival tumor tissue determined by three different anti-RB antibodies. Int. J. Cancer. 1994;58:161-167
5 Shin RW, Iwak T, Kltamoto T, et al. Hydrated aut
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