**9. Choice of detection system and concluding remarks**

The sensitivity of an IHC staining is a function of detection method for signal amplification [138].

The choice of a detection system is mainly determined by laboratories based on the nature of the specimen, expression level of the antigen, cost, desired sensitivity, and possible automation [53]. Choosing an appropriate detection system enables maximum sensitivity and optimum visibility of the immune reaction with the fewest steps and in the shortest time [139]. As a general rule, the more complex an IHC method, the more sensitive it is. One- or two-step IHC procedures are usually less sensitive than more complex, multistep procedures. In addition, the detection system must be accurate, reproducible, and results in a high signal-to-noise ratio [140]. When choosing a desirable detection system, several factors are needed to be taken into consideration: (1) the expertise/experience of the technician; (2) type of the antigen to be identified; for example, some antigens are widely expressed and do not need a sensitive method to be visualized; (3) number of tests and the amount of antibody that is available; (4) the affinity of the antibody: each antibody has its own affinity that requires a specific detection system, antibodies with less affinity usually need more sensitive detection systems; (5) species idiosyncrasies (does the tissue contain endogenous biotin), (6) budget; (7) localization of the antigen of interest (some detection systems do not have high cell penetration capacity due to the large size and regardless of having high sensitivity for detection of surface antigens, do not yield a high sensitivity for intracellular or nuclear antigens), (8) the need for or type of antigen retrieval; typically, a non-biotin-labeled detection system is recommended if HIER is used to avoid background from endogenous avidin-biotin activity (EABA) [141].

A detection system should be compatible with animal species as well. A detection system with an outstanding performance in human is not always suitable for animal models [6, 142]. The sensitivity of commercially available detection kits, some optimized for particular animal species, should be validated in-house before use. The secondary and tertiary reagents of some kits may contain antibodies or other compounds that potentially nonspecifically react with tissue antigens, leading to a background or staining. This is one justification for negative controls in IHC [141].

As a further general rule, one should always try to use the simplest detection method with sensitivity enough for detection of the antigen. The multilayering of detection antibodies beyond this threshold can be problematic as with the addition of every new step, the risk of nonspecific interaction with the preparation increases. There are some exceptions to this rule. For example, in tumor-specific antigens, which are not expressed in normal condition, the use of more sensitive methods might decrease detection level cutoff and increase the likelihood for early detection of cancer [143, 144].

In emergency conditions when results are needed in a short amount of time (such as evaluating intraoperatively surgical margins of tumor specimens), applying a detection method with high sensitivity will definitely improve accuracy of the procedure and help surgeon to obtain wider surgical margins if needed.

### **Acknowledgements**

The authors dedicate this book chapter to all mice, which generously made substantial contribution for improving authors' knowledge of immunohistochemical staining during experimental researches.

*Immunohistochemistry - The Ageless Biotechnology*
