This is part two of a two-part series on how to optimize your IHC protocols. Part one introduced the principles behind antigen retrival. Click here if you missed it... but, if you've got your tissue prepped and ready to go, we'll move on to the next steps in the staining protocol.
Step 2: Primary antibody diluent
Good IHC protocols provide the proper conditions for a specific binding event to occur. It is therefore, 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 for this purpose. It is isotonic to normal saline and buffered around physiological pH (7.2-7.6). These conditions mimic the environment that antibodies and antigens encounter during the natural course of an immunesystem interaction. As a result, TBST works well for many antibodies...but not for all. For example, PLK1 (208G4) Rabbit mAb produced limited signal when it was diluted in TBST, but the signal improved when we diluted it in SignalStain® Antibody Diluent (CST #8112)(B).
Several other proprietary diluents are available commercially and each comprises a different mixture of buffering components, detergents and/or protein stabilizers. In this case, a proprietary antibody diluent mix supported the interaction between PLK1 (208G4) and its epitope better than TBST. Since each commercially available diluent mixture is proprietary, the investigator will need to determine the suitability of each on a case-by-case basis.
Next up . . .
Step 3: 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 and the streptavidin molecule itself is conjugated to HRP. 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 (see Step 1) 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 that we had made. As a result, we switched to the more sensitive polymer-based detection method. Despite an appreciable increase in signal (C), it was not sufficient to meet our standards.
But, we had one more trick up our sleeve...
Step 4: Chromogen
Chromogens like DAB (3,3'-Diaminobenzidine), AEC (3-amino-9-ethylcarbazole) or 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 (D) that we were finally able to recommend PLK1 (208G4) Rabbit mAb for use in IHC with a defined protocol that included specific companion reagents.