Cell Signaling Technology
Scientific Writer/Editor
Cell Signaling Technology
Head, Immunohistochemistry Group
It’s well known that highly specific, high-affinity primary antibodies are the key to any successful immunohistochemistry (IHC) experiment. Perhaps less well known, companion reagents (such as buffers) that establish the pH and ionic strength of the system are similarly important. These reagents can influence the binding of the primary antibody to its epitope and dramatically affect the outcome of the assay. In fact, experiments can fail if these reagents cannot support the necessary protein-protein interactions.
Commercial antibody suppliers understand how important companion reagents are to the performance of their products, which is why they often supply companion-reagent recommendations along with their antibodies. These recommendations are a good place to start when you are planning experiments, but companies don’t generally do an exhaustive search to match their antibodies to the best companion reagents. So you may need to make adjustments if your results are not ideal. Moreover, you may wish to use more than one antibody on your sample and will need to select reagents that balance the requirements of each antibody.
Here, to help you pick the best reagents for your assay, we will review some basic principles of IHC. To illustrate our points, we will discuss how we optimized the protocol for Cell Signaling Technology’s (CST’s) PLK1 (208G4) Rabbit mAb (CST catalog #4513). When we first tested this antibody, it produced negligible signal using our standard IHC protocol (Figure 1A). By changing the companion reagents one by one, we were able to generate a strong, clean signal without changing the dilution factor of the primary antibody.
Figure 1: IHC analysis of paraffin-embedded human colon carcinoma using PLK1 (208G4) Rabbit mAb with various companion reagents, as indicated.
Heat-induced epitope retrieval
Cross-linking fixatives, like formalin, often are used to prepare samples for IHC because they preserve the structural integrity of the tissue. Although cross-linked proteins are important for maintaining tissue architecture, they may bury the epitope your antibody is designed to recognize.
There are several methods for revealing epitopes that have been masked by fixation, including proteolytic-induced antigen retrieval, which relies on an enzyme like proteinase K, or heat-induced epitope retrieval (HIER), which uses heat to break apart cross-linked bonds and unwind proteins. Either method can unmask epitopes, rendering them accessible to the primary antibody and amenable to staining by IHC. At CST, we most often use HIER when testing our antibodies, so this is the method we will discuss in detail.
HIER is performed by immersing tissue sections in a solution with a defined buffering capacity. The pH of the buffer helps keep the proteins unwound after the temperature has returned to normal, so the pH range of the system should be optimized to the antibody-epitope interaction of interest. The slightly acidic buffer citrate (pH 6.0) is effective at unmasking a wide range of epitopes, but some may require a more basic buffer, like EDTA (pH 8.0).
We used citrate buffer for HIER while optimizing the PLK1 (208G4) Rabbit mAb protocol, because it works for the widest range of epitopes. We kept this method constant as we did a step-wise change of the other companion reagents.
Primary antibody diluent
IHC depends on encouraging a specific binding event to occur. Therefore, it is essential that you calibrate the ionic strength and pH of the antibody diluent to the specific antigen-epitope interaction you are trying to detect. TBST (137 mM NaCl, 20 mM Tris, 0.1% Tween-20, pH 7.6) is commonly used as an antibody diluent, because it is isotonic to normal saline and buffered to approximately physiological pH (7.2 to 7.6). These conditions mimic the environment that antibodies and antigens encounter during the natural course of an immune-system interaction. Accordingly, these conditions work well for many antibodies, but not for all. For example, PLK1 (208G4) Rabbit mAb showed limited signal when it was diluted in TBST, but we saw increased signal when we diluted it in SignalStain® Antibody Diluent (CST #8112) (Figure 1B).
Several other proprietary diluents are also available commercially, and each comprises a different mixture of buffering components, detergents and/or protein stabilizers. In our test case, a proprietary antibody diluent mix supported the interaction between PLK1 (208G4) and its epitope better than TBST. Each commercially available diluent mixture is proprietary; therefore, the investigator will need to determine the suitability of each on a case-by-case basis.
Detection reagents
Detection reagents carry enzymes (usually horseradish peroxidase (HRP)) to the site of the specific epitope by binding to the primary antibody, either directly or indirectly through a secondary-antibody intermediate. When a chromogenic substrate for HRP is introduced, a precipitate is formed that deposits at the site of the primary antibody/antigen-binding event, making it visible upon microscopic examination.
Typically, this interaction is facilitated by biotin and streptavidin: The secondary antibody is conjugated to biotin, which can bind streptavidin. The streptavidin molecule itself is conjugated to HRP. But this method has several limitations. First, the streptavidin-HRP complex can bind to endogenous biotin and produce significant background signal. Endogenous biotin can be especially problematic if HIER is used, as buried biotin is unmasked alongside other epitopes. Second, the intensity of the chromogenic signal is determined by the amount of enzyme present (i.e., more enzyme, higher signal), but the number of HRP molecules bound to the site of the primary antibody/antigen interaction is restricted to the number of streptavidin molecules bound to the complex.
Polymer-based systems are gaining in popularity, because they avoid the limitations of the biotin-based system. In this method, a polymer, like dextran, is conjugated to a secondary antibody and the HRP enzyme simultaneously. The polymer backbone eliminates the need for building biotin-streptavidin-HRP complexes at the primary antibody/antigen site, eliminating the potential for biotin-based background noise. Plus, the number of HRP molecules that can be bound is higher relative to the streptavidin system; so fewer primary antibody/antigen-binding sites produce proportionally more signal, increasing the sensitivity of the assay.
When we used a biotin-based detection system to evaluate the PLK1 (208G4) Rabbit mAb, we found it didn’t provide a strong enough signal, even with the other changes in companion reagents we had made. Consequently, we switched to the more sensitive polymer-based detection method. Despite an appreciable increase in signal (Figure 1C), it was not sufficient to meet our standards.
Chromogen
Chromogens like DAB (3,3'-Diaminobenzidine), AEC (3-amino-9-ethylcarbazole) and Vector® NovaRED™ are substrates that interact with the HRP that is bound to the detection reagent. Several chromogenic substrates are available, and they produce a variety of colors with varying intensity. In designing your experiment, choose a chromogen that produces a color with appropriate contrast to your counterstain and of high enough intensity to reveal the antigen you are trying to detect.
Originally, we tested PLK1 (208G4) Rabbit mAb with Vector® NovaRED™ (Vector Laboratories) as our chromogen, which produces a bright red precipitate. However, as NovaRED did not provide a sufficient signal, we switched to the more intense, dark-brown DAB. The signal achieved with the DAB substrate in combination with the specific diluent and detection system was robust enough that we were finally able to recommend PLK1 (208G4) Rabbit mAb for use in IHC (Figure 1D) with a defined protocol that includes specific companion reagents.
Similar optimization should help you get the best results from your own IHC studies.
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