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17 January, 2024 by Anshul (neobio)
Immunohistochemistry (IHC) is a powerful technique used to detect and localize specific antigens in cells and tissue. This tool performs optimally for research settings, and its value in the clinical diagnostics landscape is continually increasing. An appreciation of the IHC process, compatible with formalin-fixed, paraffin-embedded (FFPE) tissue and automated methods for reproducibility, can aid in the astute selection of IHC antibodies.
IHC experiments require highly specific antibodies. These are typically targeted against certain antigens present in particular tissues and cells, which assists in determining the cellular type and origin of the organ.
In IHC, there are two primary detection protocols: direct and indirect. The direct detection method employs labeled primary antibodies to target antigens. On the other hand, indirect detection necessitates using secondary antibodies, key for signal amplification, since more than one secondary antibody molecule can bind to each primary antibody.
Both primary and secondary antibodies play integral roles in IHC. Primary antibodies target and bind directly to the antigen, while secondary antibodies recognize primary antibodies. The appropriate selection and usage of these antibodies have a profound impact on the success of your IHC experiments.
Key points about the antibodies for IHC:
When it comes to selecting the right antibodies for IHC, there are several critical factors to take into account.
The first step in choosing an antibody for IHC is defining your protein target of interest. Protein targets can be complex, given their diverse nature and structures. Thus, it is crucial to the target, its function, and its location within a cell. This information will guide the selection of the most appropriate antibody that can bind specifically and effectively to the target.
The next factor to consider is the compatibility of the antibody with your sample. Antibodies vary in their sequence and structure, which can affect their ability to recognize and bind to target proteins. This is particularly important when working with samples from different species, as the same protein can have slight variations in its sequence across species. Therefore, always ensure that the chosen antibody is compatible with the species and tissue type of your sample.
The choice between monoclonal and polyclonal antibodies is another crucial decision. Monoclonal antibodies bind to a single epitope, offering high specificity. Polyclonal antibodies, on the other hand, recognize multiple epitopes on a single target protein, offering greater sensitivity and often more stability over a range of pH and salt concentrations.
Lastly, the process of antigen retrieval (AR) is vital in IHC. AR involves the pretreatment of tissue to retrieve antigens masked by fixation, making them more accessible to antibody binding. This step significantly increases the sensitivity of IHC and expands its application. The method of AR depends on the specific target antigen and antibody. Thus, choosing an antibody that is compatible with the AR method you intend to use can greatly influence the success of your IHC experiment.
The incubation time and temperature play a significant role in the success of IHC. They influence the binding of the primary antibody to the target antigen. It’s important to find the optimal balance, as too short an incubation time may lead to insufficient binding, while too long can cause over-staining or high background signals.
Usually, a longer incubation time at a lower temperature (for instance, overnight at 4°C) is preferred over a shorter period at room temperature. However, these conditions can vary based on the specific antibody and antigen involved in your study. Thus, it’s recommended to titrate different antibody dilutions while keeping the incubation time and temperature constant to achieve the best results.
Achieving specific staining with minimal background signal is crucial for clear and accurate IHC results. If you’re obtaining specific staining but also experiencing a high background signal, you might need to adjust the incubation time and temperature again.
Additionally, consider the blocking step. This process prevents non-specific binding of the antibodies to proteins other than the target antigen, which can contribute to the background signal. The choice of the blocking agent can also influence the background, and it is worth experimenting with different agents to see which one gives the lowest background for your specific situation.
Very high dilutions may also lead to signal attenuation, especially where incubation times are limited. In such cases, antibody amplification systems can be employed to overcome this challenge. Though these systems allow a higher dilution of the primary antibody (and hence higher specificity), they can also amplify non-specific signals if not properly controlled.
IHC remains a cornerstone technique for studying protein localization within tissues, and its success hinges on the thoughtful antibody selection and protocol optimization. From defining your target protein and selecting between monoclonal or polyclonal antibodies to fine-tuning incubation conditions and controlling background staining, each step can profoundly impact the quality of your results. As antibody technologies evolve and automation becomes more integrated, mastering these fundamentals will ensure reliable, reproducible, and meaningful outcomes in both research and clinical diagnostics.
