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Immunohistochemistry Antibodies

Immunohistochemistry (IHC) is a technique that uses antibodies for detecting specific antigens in tissue samples. Unlike other common antibody-based methods, such as ELISA and flow cytometry, IHC allows for visualizing cellular components in the context of the native tissue architecture, offering critical insight into how antigen distribution and relative abundance differ under conditions of health and disease.

A typical IHC workflow begins with preserving excised tissue material, either by snap-freezing in liquid nitrogen or through formalin fixation and paraffin-embedding (FFPE). Next, the samples are sliced into thin sections and applied to glass slides, prior to blocking, immunostaining, and mounting. Besides the preservation method, other key protocol variables include whether to perform epitope retrieval, if sample permeabilization is required, and which antibodies to use.

Antibodies for IHC can be either polyclonal or monoclonal. Because polyclonal antibodies recognize multiple epitopes, their antigenicity is less likely to be affected by fixation-induced conformational changes than that of monoclonal antibodies. Polyclonal antibodies are also available from more host species than monoclonal antibodies, which can simplify multiplexing. However, polyclonal antibodies have a higher associated risk of cross-reactivity than monoclonal antibodies, increasing the potential for non-specific background signal.

Another important consideration when selecting immunohistochemistry antibodies is whether to perform direct detection (with labeled primary antibodies) or indirect detection (with labeled secondary antibodies). Direct detection involves fewer protocol steps than indirect detection, meaning faster time to results, while a major advantage of indirect detection is that it can provide signal amplification. When performing IHC with indirect detection, the use of cross-adsorbed secondary antibodies is recommended to minimize potential cross-reactivities.

Additionally, there is a need to decide between chromogenic detection (using enzyme-labeled antibodies and chromogenic substrates) or fluorescent detection. While chromogenic detection was once reserved for measuring just a single analyte, the methodology has evolved to enable multiplexed staining, albeit with a more limited dynamic range than fluorescent detection. In general, fluorescent detection is more widely used, and the simultaneous measurement of four or more targets is increasingly common.

Whichever approach is chosen, it is essential to identify antibodies that have been validated for IHC and that have proven specificity and sensitivity for their antigenic targets. The selection process should include confirming whether frozen or FFPE tissue was used for validation purposes, since an antibody that works in one sample type cannot be guaranteed to work in the other, and checking for reactivity with the intended sample species.

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Frequently Asked Questions

One of the main factors to consider when selecting a primary antibody for IHC is whether it has been validated for the IHC application. This should include establishing whether validation was performed with frozen or FFPE tissue, since antibodies cannot be assumed to work in both sample types, and seeing proof of specificity based on staining with appropriate positive and negative control samples.

Other important considerations include whether the antibody has been shown to detect the target of interest in the intended sample species and whether it is supplied as a conjugate or requires a secondary antibody for its detection.

Researchers should also weigh up the pros and cons of monoclonal versus polyclonal immunohistochemistry antibodies during product selection. While monoclonal antibodies are less likely to exhibit cross-reactivity due to their inherent monospecificity, polyclonal antibodies are generally more resilient to conformational changes in the antigen following sample fixation.

I would add a section here on the importance of selecting the best, optimal, proper, whatever description, etc. secondary antibody for use in IHC. For example, if using a mouse host antibody on rat tissue, an anti-mouse secondary antibody that has been cross-adsorbed against rat (if polyclonal; e.g., Cat. No. 1034) or an anti-mouse monoclonal secondary antibody which inherently exhibits minimal cross-reactivity with rat (e.g., Cat. No. 1144) should be used.

A major advantage of using secondary antibodies for IHC is their capacity to provide signal amplification, which can increase the likelihood of detecting low-abundance targets. However, selecting the right secondary antibody is essential to minimize the risk of cross-reactivity with the sample, which could lead to non-specific background signal.

A popular approach is to use a monoclonal secondary antibody which inherently exhibits minimal cross-reactivity to the sample species. For example, if using a mouse host IgG1 primary antibody with rat tissue, monoclonal Rat Anti-Mouse IgG1 (Cat. No. 1144), which exhibits minimal cross-reactivity against rat proteins, is a possible option. Alternatively, using a polyclonal secondary antibody that has been cross-adsorbed against the sample species is common. In the scenario just described, Goat Anti-Mouse IgG(H+L), Rat ads (Cat. No. 1034) could be used to mitigate non-specific background signal.

Another factor to consider for secondary antibody selection is the choice of conjugate. Antibodies labeled with enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (AP) are well-suited for chromogenic detection, while antibodies labeled with fluorophores have utility for immunofluorescent staining applications.

A key difference between monoclonal and polyclonal antibodies is that monoclonal antibodies recognize just a single epitope while polyclonal antibodies exhibit multi-epitope recognition. In an IHC setting, this means that monoclonal antibodies are less likely to exhibit cross-reactivity than polyclonal antibodies, but are more susceptible to epitope masking by sample fixation. This is where the formation of protein crosslinks masks the antigen, limiting antibody-epitope binding. In contrast, polyclonal antibodies have a higher potential for cross-reactivity, but epitope masking does not necessarily preclude target recognition.

While some antibodies may work with both frozen and FFPE tissue samples, this cannot be guaranteed. Researchers are advised to review the product datasheet for information about how the immunohistochemistry antibody was validated in order to identify a suitable product for their sample type.

While we do not routinely use IHC as a QC application for our secondary antibodies, if your primary antibody is validated for IHC, and you have optimized your staining conditions, it is highly likely that a secondary antibody from SouthernBiotech will work in this setting. Testing with relevant controls should be performed to confirm specific staining, and you may also wish to check the literature for protocol recommendations. Where available, literature citations are included on our antibody datasheets.

Recommended controls for IHC experiments include the following:

  • Positive expression controls – tissues expressing the target of interest are used to confirm that any staining seen in the test samples is valid
  • Negative expression controls – tissues lacking target expression are used in conjunction with positive expression controls to verify experimental results
  • Secondary antibody only controls – samples are incubated with only the secondary antibody (no primary antibody incubation) to check for non-specific secondary antibody binding
  • Isotype controls – the primary antibody is substituted for an antibody of the same isotype, but which lacks specificity for the target of interest, to identify non-specific interactions between the antibody and the sample
  • Antigen blocking controls – the primary antibody is incubated with the antigen overnight, before being applied to the sample, to confirm target specificity
  • Endogenous fluorescence controls – the sample is fixed and blocked (but not immunostained) prior to visualization with a fluorescence microscope, to determine whether endogenous fluorescence (e.g., due to collagen, riboflavin, or NADH) is present (only applicable to fluorescent detection)
  • Endogenous enzyme activity controls – the sample is incubated directly with the enzyme substrate to identify endogenous enzyme activity, such as that resulting from endogenous peroxidase or phosphatase (only applicable to chromogenic detection)
  • Antibody specificity controls – these could include testing the antibody in a different application, or comparing staining to another antibody for the same target, to increase confidence in antibody performance

With over four decades of experience in producing antibodies for scientific research, SouthernBiotech is well-placed to offer expert technical support when it comes to selecting and using antibodies for IHC. Our scientists are experienced in many different IHC applications, including the use of frozen and FFPE tissue samples for both chromogenic and fluorescent detection, and can provide the professional guidance that you need to help keep your project on track.

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