Unmasking Epitopes Using Antigen Retrieval for Formalin-Fixed Tissue Staining

 In this article, we have some tips to get you up and running with antigen retrieval to enhance your staining and alleviate your stress.

When is antigen retrieval necessary?

Formalin tissue fixation can lead to protein cross-linking or conformational changes by introducing methylene bridges, which may mask the epitope you are trying to detect and reduce primary antibody binding. Antigen retrieval reverses these processes, allowing the antibody to bind onto the antigen. If you have weak or absent staining, this can be an indicator that antigen retrieval may be required.

Antigen retrieval is a technique that unmasks an epitope, restoring the antibody’s ability to bind. It is used on tissues where antigens are cross-linked or otherwise impacted by the fixation process. Antigen retrieval is more likely to be needed when using monoclonal antibodies versus polyclonal antibodies, as polyclonal antibodies can bind to multiple epitopes whereas monoclonal antibodies only bind one. However, polyclonal antibody staining can often be improved with antigen retrieval. Either way, antigen retrieval can help you unlock the secrets hidden in your tissue with consistent staining results.

What do you need to consider when selecting antigen retrieval?

Now that you’ve taken a deep breath, accepted your fate, and decided to try antigen retrieval, there are three main considerations for protocol optimization, dependent on equipment and buffers: pH, temperature, and incubation time. Usage instructions from the primary antibody manufacturer should offer some guidelines on where to start.

1. pH — Finding the right pH can be antibody-dependent. Citrate or tris buffers are commonly used, and higher pH may lead to better unmasking. However, higher pH treatment can also cause damage to the tissue. The optimal antigen retrieval pH must be determined empirically, but it is usually around 6 (using a citrate buffer) or 9 (using a tris buffer). The optimal pH depends on multiple variables, such as the target antigen and the primary antibody clone. We recommend first checking the primary antibody manufacturer’s recommendation.
2. Temperature — Effective antigen retrieval is highly dependent on high temperature heating. The temperature is dependent on the apparatus used for antigen retrieval (water bath, pressure cooker, vegetable steamer, etc.). Since these apparatuses have different heating properties, we recommend starting low and increasing the temperature incrementally to optimize the protocol. A range of 90-100°C is generally a good starting point for testing. Be careful, however — temperatures that are too high can denature your protein!
3. Incubation Time — Test out times and increase as needed, being sure not to exceed 30 minutes. Similar to temperature, incubation time needs to be optimized for your selected apparatus. For example, 1 minute of heating at 95°C for a pressure cooker might be a good place to start.

It’s important to note that excessive heating temperature and duration can lead to tissue damage, so be sure to start low and slow, increasing your parameters incrementally — it’s all about finding the optimal middle ground to unmask your epitope without destroying your tissue!