Immunohistochemistry technology has become an indispensable and important method in the routine work and scientific research of the pathology laboratory. It is widely used in clinical pathological diagnosis to help evaluate the degree of tumor proliferation and staging, and has a guiding role in the prognosis and treatment of patients. . However, in daily work, pathological specimens are fixed with 10% neutral formalin solution. Paraffin tissues fixed with 10% neutral formalin solution will produce protein cross-linking reaction and cover certain protein antigens in the tissue. It prevents the binding of antigen and antibody. In order to open the cross-links between proteins and fully expose the blocked antigens, antigen retrieval technology has become an extremely important link in the immunohistochemical staining process. Different antigen retrieval methods directly affect the expression of immunohistochemical reaction results. The correct application of antigen retrieval technology plays an important role in immunohistochemical reaction experiments. Commonly used antigen retrieval techniques include high temperature and high pressure antigen retrieval, microwave antigen retrieval, and boiling antigen retrieval There are three methods. This article compares the three methods to discuss the effect of high temperature and high pressure antigen retrieval method on improving the results of immunohistochemical reaction.
Tissue Section Processing
Mount the paraffin tissue sections on the anti-removal glass slides and place them in a baking sheet box at 65°C overnight. The conventional tissue sections are deparaffinized in xylene. After hydration with gradient ethanol, they are rinsed 3 times with distilled water, and then placed in 3% H2O2 (hydrogen peroxide solution) for 10min to inactivate the endogenous peroxidase activity, rinsed with distilled water 3 times, washed with PBS solution for 3×3min, and pretreated 3 kinds of antigens.
Type of IHC Antigen Retrieval Method
High Temperature and High Pressure Antigen Retrieval Method
After deparaffinizing the tissue section into water, pour the 0.01mol/L citrate buffer solution (pH 6.0, antigen retrieval solution) 1000-1500mL into the electric pressure cooker, and place the tissue section on the high temperature plastic section rack. Gently put it into the electric pressure cooker, the tissue section must be under the liquid surface, close the pressure valve of the pan, plug in the power source, and adjust the timer to 180s. When the pressure of the electric pressure cooker reaches the expected value, it will be maintained for 2 minutes. After that, turn off the power and open the air jet valve. After the air deflation is complete, open the lid of the electric pressure cooker to cool to room temperature. Rinse with distilled water 3 times, and then wash with PBS solution 3 times, 3min each time. After washing, add the primary antibody diluted 1:100, incubate at 37°C for 2h, wash out the unbound antibody with PBS solution, and repeat the washing 3 times, 3 min each time. After washing is completed, add the secondary antibody, incubate at 37°C for 30 min, and then continue to wash with PBS solution for 3 times, 3 min each time. Finally, DAB was added for color development, and hematoxylin counterstaining was performed. After dehydration, the sections were sealed with gum and finally observed.
Microwave Antigen Retrieval
After the tissue section is deparaffinized and put into water, the antigen retrieval solution 0.01mol/L citrate buffer solution (pH value 6.0) 100-150mL is poured into the microwave box, and the tissue section is placed on the high-temperature-resistant plastic section rack. Put it into the microwave box, close the lid, put it in the microwave oven, first treat it on high heat for 5 minutes, and continue to treat it on medium heat for 10 minutes to keep the liquid temperature in the container at about 98 ℃. Stop the fire, take out the container, and cool to room temperature. Rinse with distilled water 3 times, and then wash with PBS solution 3 times, 3min each time. After washing, add the primary antibody diluted 1:100, incubate at 37°C for 2h, wash out the unbound antibody with PBS solution, and repeat the washing 3 times, 3 min each time. After washing is completed, add the secondary antibody, incubate at 37°C for 30 min, and then continue to wash with PBS solution for 3 times, 3 min each time. Finally, DAB was added for color development, and hematoxylin counterstaining was performed. After dehydration, the sections were sealed with gum and finally observed.
Boiling Antigen Retrieval Method
After the tissue section is deparaffinized and put into water, the antigen retrieval solution 0.01mol/L citrate buffer solution (pH value 6.0) 1000-1500ml is poured into the boiling pot, and the tissue section is placed on the high temperature plastic section rack. Gently put it into the boiling pot, the tissue section must be below the liquid surface, count for 30 minutes after boiling, turn off the heat, and cool to room temperature. Rinse with distilled water 3 times, and then wash with PBS solution 3 times, 3min each time. After washing, add the primary antibody diluted 1:100, incubate at 37°C for 2h, wash out the unbound antibody with PBS solution, and repeat the washing 3 times, 3 min each time. After washing is completed, add the secondary antibody, incubate at 37°C for 30 min, and then continue to wash with PBS solution for 3 times, 3 min each time. Finally, DAB was added for color development, and hematoxylin counterstaining was performed. After dehydration, the sections were sealed with gum and finally observed.
The heated antigen retrieval (AR) technology is an important milestone in the history of immunohistochemistry. The principles of the three types of antigen retrieval are roughly the same. Through the action of high-frequency electromagnetic waves or high temperature and high pressure, the aldehydes formed by the treatment of the cross-linkable fixative formaldehyde in the tissue The chain opens to fully expose the antigen and achieve the purpose of repair. By comparing the pretreatment of three different antigen retrieval methods, it is found that the pretreatment using the high temperature and high pressure antigen retrieval method is significantly better than microwave retrieval. Because high temperature and high pressure can overcome batch differences and “hot spots” in the use of microwave ovens, it is believed that high temperature and high pressure are more uniform than other heating methods. When the electric pressure cooker is fully pressured at 15 Psi, the temperature can reach about 120 ℃. This kind of temperature shows obvious superiority in the de-masking effect of some nuclear antigens. More antigens can be exposed, and some false negative and weakly positive specimens can be positively expressed. The positive location of the tissue is clear, the background color is light, and the non-specific coloration is weak. It can improve the positive detection rate of antigen and antibody, simple operation and short time consumption. The positive expression rate is stronger than the microwave antigen retrieval method; while the microwave antigen retrieval method, due to the narrow heating range of the microwave oven, the tissue slices receive the microwave radiation unevenly and the amount is small, resulting in the inconsistency of the processing intensity of the tissue slices in different regions. Edge effect, resulting in the appearance of different shades of color in different areas. At the same time, microwave oven heat repair is prone to the phenomenon of dry slices caused by dry buffer solution, and many disadvantages such as longer repair time and easy tissue peeling; while boiling antigen retrieval, the antigen retrieval time is long, the temperature is not easy to control, and it is easy to heat unevenly can lead to poor recovery of the antigen activity of the tissues, thereby affecting the results of immunohistochemical reactions. Therefore, the high temperature and high pressure antigen retrieval method is significantly better than the microwave antigen retrieval method and the boiling antigen retrieval method.