The most important factor when designing an IHC/ICC experiment is selection of the primary antibody. In turn, the critical feature of a primary antibody is specificity for the epitope. All steps of an IHC/ICC experiment must be optimized to visualize specific staining and minimize non-specific background signals. This includes performing initial studies to determine the appropriate incubation conditions for each primary antibody. The working dilution for an antigen affinity-purified polyclonal antibody is generally lower than that of a monoclonal antibody but these values must be determined empirically. To achieve a robust and specific signal, a high quality antibody that exhibits minimal cross-reactivity should be employed.
Starting conditions for Primary Antibody Incubation
||5-25 µg/mL, overnight at 4 °C
||1.7-15 µg/mL, overnight at 4 °C
||5-25 µg/mL, 1 hour at room temperature
||1.7-15 µg/mL, 1 hour at room temperature
||Single epitope specificity
||Lower concentration required
||Vulnerable to epitope masking
Selecting a Primary Antibody Supplier
When searching for a primary antibody, it is likely that products will be available from several different commercial sources that may offer varying degrees of antibody quality. This initial choice can make the difference between a successful experiment and a missed opportunity. A first step is to look for independent verification. This includes checking for prior successful use of the antibody in the literature. Many commercial suppliers, including R&D Systems, can provide assistance to researchers looking for this information. Another potential question to ask is whether the antibody is actually manufactured by the supplier or is it purchased and resold? Purchasing direct ensures the commercial source has had complete control of manufacturing, quality control processes, and shipping and storage conditions. In addition, the manufacturer may be in the best position to provide quality technical service should questions arise about the use of the antibody.
Selecting a Monoclonal or Polyclonal Antibody
| Monoclonal and Polyclonal Antibody Binding. Monoclonal antibodies bind only a single epitope while polyclonal antibodies bind different epitopes on the same protein.
The inherent features of monoclonal and polyclonal antibodies are associated with advantages and limitations when applied to IHC/ICC. Monoclonal antibodies, produced from a single B cell clone, represent a homogeneous population that bind with high affinity and specificity to a single epitope. This can be particularly useful when detecting a single member of a protein family that shares a high percentage of amino acid identity.
Antibody binding is often dependent on the target protein maintaining its native conformational state. Access to a desired epitope can be compromised by interactions with other proteins, post-translational modifications, temperature, pH, fixation, and salt concentration. Because polyclonal antibodies are heterogeneous and can recognize multiple epitopes, they are less likely to be affected by changes in protein conformation. In general, polyclonal antibodies are also more stable than monoclonal antibodies over a range of pH and salt concentration. For these reasons, polyclonal antibodies are more frequently used for IHC/ICC experiments than monoclonal antibodies.
Affinity Purification of Polyclonal Antibodies
Polyclonal antiserum contains a mixture of antibodies produced by a large number of B cell clones. Antibodies comprising polyclonal antiserum bind to target epitopes with different specificities and affinities, as well as cross-react with irrelevant target molecules (non-specific interactions). To enrich for antibodies with the highest specificity and affinity toward the antigen of interest, R&D Systems polyclonal antibodies are antigen affinity purified. During this process polyclonal antiserum is passed through an affinity column containing immobilized antigen molecules. Specific antibodies are retained by the immobilized antigen, whereas non-specific antibodies pass through the column and are discarded. The retained antigen affinity purified antibodies are subsequently eluted from the column. Antigen affinity purified antibodies interact predominantly with their target antigen, reduce background staining, and produce more consistent results compared to non-purified antibodies.
Optimization of Primary Antibody Incubation
Primary antibody concentration, diluent, incubation time, and temperature all impact the quality of staining. These variables need to be optimized for each antibody and sample to achieve specific staining and low background. Often optimization is approached by maintaining a constant incubation time and temperature, while varying the antibody concentration to determine when an optimal signal is achieved with low background noise. For example, if a high affinity antibody is used, then the antibody can be used at a relatively high concentration for a shorter incubation time. Alternatively, a high affinity antibody at lower antibody concentration may be used with a longer incubation time. Longer incubation durations are often employed to ensure penetration throughout tissue sections used for stereological techniques. To promote specific staining, longer incubation periods are often conducted at lower temperatures (i.e. 4 °C versus room temperature).
Optimization of R&D Systems staining protocols for tissue sections typically begin with an overnight incubation with the primary antibody at 4 °C. For staining cells, a 1 hour incubation with the primary antibody at room temperature is a common starting point. The working dilution of an antigen affinity purified polyclonal antibody is generally lower (from 1.7-15 µg/mL) than that of a monoclonal antibody (5-25 µg/mL). When comparing samples stained with different concentrations of the same antibody, it is important to use a consistent time and temperature for the incubation step. When working with a new antibody for the first time, it is recommended to conduct preliminary studies testing a broad range of antibody concentrations.