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How to use antigen search technology to restore protein visualization capabilities?
With the continuous advancement of science and technology, the visualization technology of protein has become one of the indispensable methods in biomedical research.In particular, immunohistochemistry (IHC) technology allows researchers to recognize and localize proteins in biological tissues through the binding of specific antibodies to antigens.This technology is of great significance for diagnosing abnormal cells, such as cancerous tissue.However, the visualization process of proteins is often obscured by antigens during immobilization and processing.At this time, the introduction of antigen search technology is particularly important.
The necessity of antigen search technology
Antigen search techniques are designed for structural changes that occur during the fixation of specimens, which can lead to masking of antigen sites.Usually, fixed specimens will produce cross-linking reactions through fixatives such as formaldehyde, causing obscurity of antigen epitopes.By using antigen search technology, we can restore these hidden antigens, thereby improving the visualization of proteins.
The most common method for antigen retrieval is high temperature treatment, usually soaking sections in buffer, which can destroy the crosslinked structure and restore accessibility to the antigen site.
Sample preparation process
When performing immunohistochemistry, a series of sample preparation steps are required, which are crucial to the final staining result.The basic process usually includes fixation, sectioning, antigen retrieval, and incubation with antibodies, etc.
Fixation and Slicing
Fixing is a key step in maintaining tissue morphology, and 10% neutral buffered formaldehyde is usually used as the fixative.After fixation is completed, the sample needs to be sliced, usually with a thickness of 4-6 microns, which can ensure sufficient antigen presentation.At the same time, paraffin must be removed before performing sectioning.
Special methods for antigen search
Antigen search techniques usually rely on physical or chemical methods to destroy fixed and crosslinked structures.There are many ways to treat high temperatures, and common techniques include microwave heating, water baths, and pressurized steam.Antigen search can also further improve signal strength for sections fixed with ethanol or acetone.
Treatment against nonspecific antibody binding is also crucial, and background staining can be effectively reduced by using normal serum or professional blocking buffer.
Dyeing and Visualization
After the sample preparation process is completed, the next step is to perform sample staining.This process usually requires the use of antibodies labeled with small molecule reporters or enzymes to specifically label the antigen of interest.These markers can make the antigen visible under a microscope.Depending on different detection methods, the antibody can be monoclonal or polyclonal, the former usually targeting a single epitope of a specific antigen, and the latter can recognize multiple epitopes.
Selecting detection method
The five main detection methods include direct detection and indirect detection.Direct detection is relatively simple and can be performed with just one labeled antibody.However, the indirect detection method is more sensitive because it allows multiple labeled secondary antibodies to bind to primary antibodies, and this signal amplification effect improves the accuracy of the detection.
Test and Calculation Analysis
After the staining, it is necessary to observe antigen performance under a microscope.The use of staining indicators such as scanning method (EDX) can perform semiqualitative and quantitative analysis of signal intensity, making the comparison of protein expression patterns more obvious.
Map of protein expression
Researchers can use immunohistochemistry to map protein expression in normal and lesion tissues.This technology combines tissue microarray technology and can effectively display protein expression patterns in different tissue types.The Human Protein Map Database provides an excellent platform to help researchers understand how proteins are distributed in different tissues.
Due to the importance of immunohistochemistry in clinicopathology, it can not only help diagnose cancer, but also provide an important basis for subsequent treatment.Against this background, we can’t help but think about what new technologies will be available in the future that will allow us to have a deeper understanding of the functions and functions of proteins?
