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23 January, 2024 by Anshul (neobio)
Snapshot of IHC and Primary Antibodies:
Immunohistochemistry (IHC) is a fascinating intersection of anatomy, immunology, and biochemistry. It offers an in-depth examination of the hidden, microscopic world of cellular structures and proteins. Primary antibodies are the lead actors in this intricate play, directly binding to specific proteins or antigens within cells, not mere points of reference, but critical plot points that reinforce the narrative of our scientific understanding.
Whether your research involves studying specific isoforms of a protein or post-translational modifications, the choice of your primary antibody is critical. The right antibody not only has to work in IHC but also must be specific enough to recognize and bind to your target protein.
Furthermore, the realm of IHC encompasses both direct and indirect applications of these antibodies. While the direct method involves the application of labeled primary antibodies to the tissue, the indirect method utilizes labeled secondary antibodies. Each comes with its own merits and limitations, shedding light on different aspects of the cellular ensemble.
Aimed at providing a comprehensive understanding of these nuanced mechanics, this article discusses topics ranging from the role of IHC in detecting proteins and antigens to the crucial aspects of primary antibodies. The specifics of direct and indirect applications of antibodies in IHC are also presented.
IHC experiments begin with meticulous tissue preparation that can significantly influence the accuracy and reliability of the results.
Proper fixation of the tissue sample is crucial for maintaining the cellular and tissue architecture while preserving the antigens. The most common fixative used is formalin, which cross-links proteins and nucleic acids in the tissue.
After fixation, the tissue is embedded in a medium, such as paraffin, which provides the structural support necessary for thin sectioning. These thin sections are then mounted on slides for subsequent staining.
Following fixation, some antigens may become masked or altered, making them less accessible to antibody binding. There are antigen retrieval (AR) techniques that help retrieve these masked antigens, making them more accessible for antibody binding.
Each AR method is suited to a specific target antigen and antibody. The most popular method is heat-induced AR (HIAR), which involves the breaking of protein cross-links caused by formalin fixation. HIAR can be achieved using various heat sources such as microwave ovens, pressure cookers, autoclaves, and water baths.
Additionally, proteolytic enzyme digestion is a technique that utilizes enzymes to cleave proteins and expose their antigenic sites. This AR method is particularly useful for epitopes that may lose their antigenicity when exposed to heat.
It is important to note that there is no one-size-fits-all approach to AR. The method chosen largely depends on the specific target antigen and antibody. Hence, it’s vital to understand the characteristics of the primary antibody and the target antigen for successful AR.
Selecting the appropriate primary antibody is crucial to maximizing the benefits of your IHC experiments. Primary antibodies are the workhorses of IHC, directly binding to the specific protein or other antigen in the tissue being studied. Thus, the type of primary antibody used can greatly influence your IHC results.
Monoclonal antibodies, as the name suggests, are derived from a single B cell clone and hence, recognize a single epitope. The specificity of monoclonal antibodies is their biggest advantage, as they are less likely to cross-react with other proteins, resulting in less background staining. This high specificity is ideal for detecting unique epitopes and reducing the chance of false positives. However, a potential limitation is that if the specific epitope is not accessible or is altered in some way, the antibody may not bind effectively.
On the other hand, polyclonal antibodies are a mixture of antibodies that recognize multiple epitopes on a single antigen. This diversity allows them to tolerate changes in the antigen’s conformation, making them more flexible and versatile. They are also more stable over a range of pH and salt concentrations than monoclonal antibodies. However, their higher chance of cross-reactivity can lead to higher background staining, which could interfere with the interpretation of results.
When choosing an IHC primary antibody, you should consider the following factors:
It is also important to mention some advanced IHC techniques that can further enhance your research efforts.
The multi-color immunohistochemistry (mIHC) technique facilitates the detection of multiple markers in a single tissue section, making it a powerful tool for generating high-content data. It enables us to better understand the relationship between different markers while reducing the amount of tissue required for analysis.
Traditional chromogenic mIHC relies on each antibody being raised in a different species or of a different isotype. However, distinguishing more than two chromogens on a slide can be challenging, especially when the spectra overlap. On the other hand, fluorescent mIHC can distinguish three or more markers using dye-conjugated secondary antibodies due to their extra amplification.
Another key aspect of IHC is the use of counterstains to highlight specific morphologies or structures. This approach facilitates the localization of primary antibodies. The most popular counterstain used with chromogenic IHC staining is hematoxylin, which stains nuclei blue, contrasting with the brown of HRP-DAB. In fluorescent IHC, the most popular counterstain is the blue nuclear dye DAPI. The choice of counterstain should be compatible with your staining system and should not interfere with the signals from your IHC primary antibodies.
Regarding detection methods, chromogens offer the advantage of compatibility with organic mounting media, resulting in sharper images. However, aqueous media are quicker with no need to dehydrate the section. On the other hand, fluorescent detection methods can simultaneously identify multiple (>3) markers.
Advanced IHC techniques, such as multi-color IHC and the use of counterstains, can significantly enhance your IHC results. By correctly applying these methods and selecting the appropriate detection method, you can ensure high-quality, accurate results from your IHC experiments.
Importantly, considering blocking to prevent non-specific antibody binding, using controls to validate staining patterns, and utilizing secondary antibodies for indirect detection and signal amplification are also crucial to ensuring the success of IHC experiments.
Notably, blocking is a crucial step in IHC. Non-specific antibody binding can lead to false positives and background staining. It involves adding a solution to the tissue to prevent non-specific protein binding. A common blocking method utilizes non-immune serum from the same animal species as the secondary antibody, thereby enhancing the specificity of the antibody–antigen interaction and reducing false-positive staining caused by non-specific protein binding.
It is also essential to run both positive and negative controls as a quality control step for IHC experiments. Positive controls involve tissues that contain an antigen known to be stained by a specific antibody. They should ideally be run on the same slide as the test tissue to undergo the same reaction conditions. In contrast, negative controls involve the test tissue undergoing the same staining conditions, but without the addition of the primary antibody. This control helps eliminate the possibility of non-specific antibody binding with the secondary antibody. Indeed, controls play a crucial role in reducing false positives and negatives that can arise due to various factors.
Secondary antibodies play a vital role in the indirect method of IHC. The primary antibody, which is specific to the antigen of interest, is not labeled in this method. Instead, a labeled secondary antibody that binds to the primary antibody is used. This approach provides signal amplification, enhancing sensitivity, and can be used with many different primary antibodies. Various labels can be used, such as fluorescent molecules and enzymes like horseradish peroxidase or alkaline phosphatase. These produce a colored product after incubation with a chromogenic substrate, allowing for the visualization of antigen–antibody binding.
Furthermore, antibody validation ensures the reliability and reproducibility of your IHC results. Make sure you use monoclonal antibodies that are validated for IHC. These antibodies typically undergo rigorous validation processes, ensuring that they recognize the intended target with high specificity and sensitivity.
In conclusion, selecting the right primary antibody for IHC is a crucial step that can significantly impact the quality of your results. By considering the type of antibody, its specificity, the host species, and its validation status, you can increase the likelihood of successful and reproducible IHC experiments.
