Beginner’s Guide to Immunohistochemistry 1: Choosing a Primary Antibody

As with any other experimental setup, there are many factors to consider when embarking on a new immunohistochemistry (IHC) project, for example, the kind of tissue type you will stain and whether it will be fixed or frozen, the labelling/detection system, suitable staining controls, and the way in which you will analyse your data, to name a few.

One of the most important factors to consider when planning an IHC experiment is the choice of primary antibody. Getting this part right is crucial for high target specificity and minimal off-target staining, and increases your chances of obtaining a consistently reliable signal that provides valid biological information about your sample. To simplify this task, we present our beginner's guide to choosing a primary antibody for a new IHC project.

Don't Reinvent the Wheel -
Go to Known Antibody Suppliers!   

In many cases, a colleague or collaborator can start you off by suggesting their go-to antibody suppliers. At Nordic BioSite, you can search here for antibodies against your target protein. Check any corresponding datasheets for validation data including staining images - has the antibody been validated by the supplier for your chosen application and sample type, e.g., IHC on frozen mouse brain tissue? 

What Antibody Is Everybody Else Using? 

If you've checked all the antibody suppliers you know and you're no further on, it's time to consult the literature! Many suppliers include citation lists for each of their antibodies independently of application, allowing you to identify the antibodies that are most widely used for your target protein. If not, you can search the literature yourself. This approach may help you identify other suppliers, as well as researchers that have used a given antibody in IHC even though the supplier hasn't listed IHC as a tested application.

Make sure you study any citations critically and that they provide enough information to you to proceed in the lab. For instance, while publications often state the catalogue numbers of antibodies used, they seldom state the antibody dilutions or staining conditions used. If this happens, reach out to the authors and ask for more information.

How Was the Antibody Validated?

Whether you identify a candidate antibody via a supplier or the literature, you'll need to put your critical hat on when it comes to validation.

If a primary antibody was validated against an overexpressed form of the target protein with good staining results in IHC, it doesn't guarantee good results when you try to stain the native protein. The same goes for antibodies that are validated in tissues that highly express your target protein - this level of validation may not be enough when looking at a low-expressing tissue type or diseased tissue where the target protein's expression level is reduced.

Some suppliers will state that an antibody has been validated for a given application without showing data or disclosing details of how the validation was carried out. If you're not convinced by the validation data shown, don't be afraid to contact the supplier and make enquiries. They may have additional information besides what's recorded on the datasheet, and if you're lucky they might provide you with a low-volume test size at a low cost or even a free sample so you can test the antibody for yourself!

Monoclonal or Polyclonal?

By this stage you might have found an antibody to start testing, or perhaps you have a few to choose from. This section deals with how you might choose between a monoclonal or polyclonal antibody if you have the option of both. 

Because of their robustness to changes in staining conditions, e.g., pH, buffer composition, tissue type, polyclonal antibodies often yield better staining results in IHC than a monoclonal antibody directed against the same antigen. Additional advantages of polyclonal antibodies for IHC include:

  • They recognise multiple epitopes on the target antigen, thus increasing their overall affinity for the target.
  • Recognition of multiple epitopes increases sensitivity, which may be especially useful for the detection of weakly expressed target proteins. 

  • They may be superior in the detection of native proteins because they recognise multiple epitopes. So, if a pretreatment, pH or temperature condition has caused some epitopes to be hidden or destroyed, other epitopes will still be exposed available for binding by a pool of the polyclonal antibody.

Before you rush off to order a stash of polyclonal primary antibodies for IHC, you should bear in mind that their ability to recognise multiple epitopes may also be a disadvantage, particularly if that makes them more likely to recognize an off-target antigen. For instance, if you are attempting to stain a protein that belongs to a family with very similar members, you may want to consider using a monoclonal primary antibody, since their specificity towards a single epitope on your target protein will greatly reduce the risk of off-target binding. However, to be on the safe side, it is strongly advisable to ensure that even a monoclonal antibody is validated for cross reactivity towards other family members. If you do take this route, bear in mind that monoclonal antibodies are often very sensitive to even slight changes in testing conditions, so you will need to develop very standardised protocols for consistent staining results. 

You've Found Your Antibody - Now What?

 You may have found an excellent primary antibody candidate, but most often you won't know if it's the right one until you go to the lab and do some staining! The optimal staining conditions for any primary antibody will depend on the experiment at hand and will therefore require a certain amount of optimisation for high quality staining. As a rule of thumb, we suggest starting out with the supplier's recommendations for staining conditions, e.g., working dilution, pretreatment conditions, incubation temperature, and blocking and washing buffer compositions. Later in this article series, we will provide tips to help you to optimise and troubleshoot IHC when staining doesn't go as planned. 

Still Looking - Get in Touch!

If you're still looking for a primary antibody and/or would like some advice on antibody optimisation or any other aspect of IHC, get in touch with us on info@nordicbiosite.com and one of our experienced team members will happily help you!

Do stay tuned for future posts covering secondary antibodies, staining controls and detection strategies for IHC!