Immunohistochemistry (IHC) assay - inactivation.
Immunohistochemistry (IHC) assays are a commonly used biological technique to detect the expression levels of specific proteins in cells or tissues. In IHC experiments, inactivation is often required to eliminate the influence of endogenous enzymes and active substances on the experimental results. This article will introduce the methods and functions of inactivation treatment in IHC experiments.
1. The role of inactivation treatment in IHC experiments.
Inactivation is one of the most critical steps in IHC experiments. Through inactivation, the effects of endogenous enzymes and active substances in cells or tissues can be eliminated, improving the specificity and sensitivity of the experiment. Common endogenous enzymes and active substances include endogenous peroxidases, phenol oxidases, and ribozymes. These enzymes and active substances may interfere with the labeled signal in IHC experiments, resulting in non-specific staining or increased background noise. Therefore, the influence of these interferences on the experimental results can be effectively reduced by inactivation.
2. Methods of inactivation treatment in IHC experiments.
1.Boiling method: The boiling method is a commonly used inactivation treatment method. Cells or tissue samples can be processed in a high-temperature and high-pressure environment to effectively inactivate endogenous enzymes and active substances. This is done by placing a cell or tissue sample in a test tube, adding an appropriate amount of water or buffer, and then placing the tube in a pressure cooker and boiling for a certain amount of time. After boiling, the endogenous enzymes and active substances in the cells or tissues are destroyed, reducing background noise and increasing the specificity of the experiment.
2.Enzyme inhibitor method: Enzyme inhibitor method is a method of inhibiting endogenous enzymes and active substances by adding specific enzyme inhibitors. Common enzyme inhibitors include phenylmethylsulfonyl fluoride (PMSF), aprotinin, and pepsin inhibitors. These inhibitors can inhibit the activity of endogenous enzymes and active substances, thereby reducing background noise and increasing the specificity of the experiment. This is done by adding an appropriate amount of enzyme inhibitor to the processing solution of a cell or tissue sample and then following the protocol.
3.Chemical reagent method: The chemical reagent method is a method of inactivating endogenous enzymes and active substances by adding chemical reagents. Common chemical reagents include methanol, ethanol, acetone, formaldehyde, etc. These chemical agents can react with endogenous enzymes and active substances, thereby destroying their activity. This is done by adding an appropriate amount of chemical reagent to the processing solution of the cell or tissue sample and then following the experimental procedure.
3. Precautions for inactivation treatment in IHC experiments.
1.When choosing an inactivation method, it should be based on the type of cell or tissue and the specific requirements of the experiment. Different cells or tissues and different experimental requirements may require different inactivation treatments.
2.When performing the inactivation treatment, attention should be paid to controlling parameters such as temperature, time, and pH. These parameters may affect the inactivation effect and experimental results.
3.When performing IHC experiments, care should be taken to follow the principle of aseptic operation to avoid the influence of sample contamination on the experimental results.
4.When performing inactivation treatment, attention should be paid to safety issues. For example, when using the boiling method, care should be taken to prevent the occurrence of safety accidents such as pressure cookers**. When using the chemical reagent method, attention should be paid to selecting the appropriate chemical reagent and concentration to avoid causing harm to the human body and pollution to the environment.
In conclusion, inactivation in immunohistochemistry (IHC) experiments is one of the most critical steps. By selecting the appropriate inactivation method and controlling the relevant parameters, the specificity and sensitivity of the experiment can be effectively improved, and accurate and reliable data support can be provided for scientific research.