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The wonderful combination of antibodies and antigens: How does immunohistochemistry work?
Immunohistochemistry (IHC) is a technique that relies on the binding of antibodies to specific antigens, through which the presence of antigens can be confirmed in cells and tissues. This method originated from the immunofluorescence technology developed by scientists such as Albert Hewett Coons in 1941, which laid the foundation for subsequent immunohistochemistry. Over time, immunohistochemistry has been widely used in the diagnosis of cancer, particularly in identifying tumor antigens. Therefore, understanding the procedures of immunohistochemistry is crucial for medical and biological research.
Nowadays, immunohistochemistry is regarded as a powerful biomarker exploration tool. Through this technology, researchers can determine the expression and localization of different proteins in biological tissues.
Sample preparation
Immunohistochemistry is performed using tissue samples that are fixed and embedded in paraffin, and sometimes cryopreserved (frozen) tissue is also used. The sample preparation process includes fixation, antigen retrieval, incubation with primary antibodies, and finally incubation with secondary antibodies. Throughout this process, every step is critical to the final outcome.
Tissue preparation and fixation
Before performing immunohistochemistry, the tissue is fixed to preserve the morphological structure of the tissue. The reagent used for fixation is usually 10% neutral buffered formalin. The fixation time and reagent ratio will significantly affect the experimental results. Generally speaking, the fixation time is 24 hours, and the ratio of fixative to tissue ranges from 1:1 to 1:20.
Slice
Sectioning of samples was performed using a microtome. Paraffin-embedded tissue is typically sectioned at a thickness of 4 microns, while cryosections are sectioned at 4 to 6 microns. The thickness of the sections is a critical factor in the success of immunohistochemistry, as differences in thickness can affect the observed antigen presentation.
Antigen retrieval
Antigen retrieval is necessary for most fixed tissue sections. Its purpose is to restore the cross-links formed during fixation to make the epitope accessible to the antibody. A common method of antigen retrieval is to immerse sections in buffer by heating at high temperatures.
The success of antigen retrieval often determines the sensitivity and specificity of immunohistochemistry testing.
Block
In immunohistochemistry, nonspecific binding of antibodies can cause background staining problems. To reduce background staining, samples are often incubated with normal serum from the species from which the secondary antibody was produced.
Sample tag
After sample preparation is complete, it can be labeled with antibodies labeled with fluorescent compounds, metals, or enzymes. Antibodies can be polyclonal or monoclonal, depending on the desired application.
Detection method
There are two main detection methods in immunohistochemistry: direct method and indirect method. The direct method uses labeled antibodies to react directly with the antigens in the sections, while the indirect method is used to improve sensitivity and increase the signal amplification effect through the combination of secondary antibodies.
The advantage of the indirect method is that it requires the generation of a relatively small number of labeled secondary antibodies, which is very useful when multiple labeling.
Reporter
The reporter molecules used in various detection methods are different, and the common ones are chromogen and fluorescence detection. In chromogen immunohistochemistry, antibodies are combined with enzymes and react with substrates to produce a visible color. Fluorescence detection uses fluorescent pigments to make the antibody glow for easy observation.
Reverse counting staining
After immunohistochemical staining is completed, reverse counting staining is often applied to provide contrast and help guide and visualize the tissue.
In many cases, reverse counting staining can significantly improve the researcher's observational capabilities.
Troubleshooting
In the application of immunohistochemistry, problems such as excessive background staining, weak target antigen staining, and artifacts may occur, which will affect the test results. Therefore, immunohistochemistry techniques should be optimized and antibody quality ensured.
Immunohistochemical markers in clinical diagnosis
This technology has become an important tool in neuroscience research, not only for confirming protein expression, but also for tumor identification. For example, CD15 and CD30 markers are used for Hodgkin's disease, while PSA is used for prostate cancer diagnosis.
Guided Therapy
As cancer treatments advance, immunohistochemistry can be used to assess which tumors are likely to respond to treatment by detecting the presence of molecular targets. Hormone receptors in certain tumors can be confirmed by immunohistochemical signals, which can help enhance the implementation of personalized therapy.
Therefore, immunohistochemistry is not only a diagnostic tool but also a key component in the clinical treatment process.
In summary, immunohistochemistry provides us with a powerful tool to identify and localize biomarkers at the cellular and tissue levels. However, how can we further optimize this technology to better serve the medical and research communities in the future?
