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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 us an in-depth look at 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.
Further, the realm of IHC involves both direct and indirect application of these antibodies. While the direct method involves labeled primary antibodies applied to the tissue, the indirect method uses 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, our journey will cover facets ranging from the role of IHC in detecting proteins and antigens to the crucial aspects of primary antibodies. We’ll also delve into the specifics of direct and indirect applications of antibodies in IHC. Let’s turn the page, and dive right in.
The journey of Immunohistochemistry (IHC) begins with meticulous tissue preparation. It’s a critical first step in the process that can significantly influence the accuracy and reliability of the results.
In IHC, the tissue sample is the canvas on which we paint our molecular masterpiece. The quality of this ‘canvas’ is paramount. Proper fixation of the tissue sample is essential to maintain the architecture of the cells and tissues 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 like paraffin, which provides the structural support needed for thin sectioning. These thin sections are then mounted on slides for subsequent staining.
Even with careful fixation, some antigens may become masked or altered, making them less accessible to antibody binding. This is where antigen retrieval (AR) comes into play. AR is a technique that helps retrieve these masked antigens and make them more accessible for antibody binding.
There are several methods of AR, each suited to a specific target antigen and antibody. The most popular method is heat-induced antigen retrieval (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.
Another form of AR is proteolytic enzyme digestion, which involves the use of enzymes to cleave proteins and expose the antigenic sites. This method is particularly useful for epitopes that may lose their antigenicity with heat.
However, it’s important to note that there isn’t a 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.
In short, the art of IHC lies in the careful preparation of the tissue and the adept retrieval of antigens. The mastery of these techniques can significantly improve the sensitivity and specificity of IHC, helping researchers to uncover the secrets hidden within their tissue samples.
To get the most out of your IHC experiments, the choice of primary antibody is crucial. Primary antibodies are the workhorse of IHC, directly binding to the specific protein or other antigen in the tissue that you’re studying. The type of primary antibody used can greatly influence the results of your IHC.
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:
Continuing on from selecting the right primary antibody, let’s dive into some advanced IHC techniques that can further enhance your research efforts.
One such technique is multi-color immunohistochemistry (mIHC). This method allows for 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, all while reducing the amount of tissue required for the 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 if any chromogens overlay each other. That’s where fluorescent mIHC comes in. This method can easily distinguish three or more markers and is commonly used with dye-conjugated secondary antibodies due to their extra amplification.
Another key aspect of IHC is the use of counterstains. These are used to highlight specific morphologies or structures, aiding in the localization of your primary antibody. 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 shouldn’t interfere with your IHC primary antibody signals.
Finally, let’s talk about detection methods. Chromogens have the advantage of compatibility with an organic mounting medium, resulting in sharper images. However, aqueous mediums are quicker to use as there’s no need to dehydrate the section. On the other hand, fluorescent detection methods can identify three or more markers simultaneously, a feat not easily achieved with chromogens.
In conclusion, advanced IHC techniques like 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.
When performing IHC, it’s important to take steps to ensure the success of your experiments. These steps include blocking to prevent non-specific antibody binding, using controls to validate staining patterns, and utilizing secondary antibodies for indirect detection and signal amplification.
In Immunohistochemistry (IHC), blocking is a crucial step. Non-specific antibody binding can lead to false positives and background staining. This is where blocking comes into play. It involves adding a solution to the tissue to prevent non-specific protein binding. A common method for blocking involves using nonimmune serum from the same animal species as the secondary antibody. This step is important in enhancing the specificity of the antibody-antigen interaction and reducing false positive staining caused by nonspecific protein binding.
Quality control is a critical aspect of IHC. With each run, it’s important to carry out both positive and negative controls. Positive controls involve tissues that contain an antigen known to stain with a certain antibody. They should ideally be run on the same slide as the test tissue to undergo the same reaction conditions. Negative controls, on the other hand, involve the test tissue undergoing the identical staining conditions minus the primary antibody. This helps eliminate the possibility of nonspecific antibody binding with the secondary antibody. 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 the primary antibody is used. This method 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, making it possible to visualize the antigen-antibody binding.
Antibody validation is a critical step in ensuring the reliability and reproducibility of your IHC results. At NeoBiotechnologies, we manufacture over 1,000 highly validated, monospecific Rabbit Recombinant Monoclonal Antibodies, ideal for Immunohistochemistry, Flow Cytometry, Western Blotting, or Immunofluorescence. These antibodies 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.
