**2. Direct method**

Direct detection methods are known as a one-step process applying a primary antibody, which is directly labeled with reporter molecules, such as biotin, colloidal gold, fluorochromes, or enzymes [1, 2]. The conjugated antibody makes a direct contact with cognate antigen in histological or cytological preparations (**Figure 1**). Direct detection methods are widely used for detecting highly expressed antigens. Furthermore, when the use of the secondary antibodies causes nonspecific and unwanted reactions, owing to the histological nature of the tissue and/or host species of the primary antibody, direct detection could be the technique of choice. For instance, in case of mouse lymph node immunostaining, labeled primary mouse monoclonal antibodies are preferred because antimouse secondary antibodies are not only bound to mouse primary antibodies bound to the tissue antigens of interest but will also react with endogenous immunoglobulins vastly found in lymph nodes. This would lead to a strong nonspecific staining. Hence, direct detection methods using mouse primary antibodies conjugated to a fluorophore or enzyme would be a better option [3]. If this approach is not practically feasible, for example, due to the low expression level of the target antigen or technical problems in primary antibody labeling, indirect methods using primary antibodies from species other than that of target tissue would be desirable.

One of the advantages of direct detection is that the incubation step with a secondary reagent is eliminated. Hence, this method is time saving and easy to

**5**

**Figure 2.**

*Indirect immunostaining method.*

*Detection Systems in Immunohistochemistry DOI: http://dx.doi.org/10.5772/intechopen.82072*

ties are produced against an antigen [5–7].

the limitations mentioned above.

**3. Indirect method**

perform. In addition, due to the wide range of fluorochromes that are commercially available, direct detection is vastly used in multicolor experimental designs [4, 5]. It is important to note that insufficient sensitivity to detect most of the antigens found in routinely processed tissues is one of the drawbacks of using direct detection method. Furthermore, each primary antibody needs to be individually conjugated with fluorophores or enzymes, which increases considerably the cost of the whole process. Another concern with direct staining methods is the possibility of functional impairment of the antibody affinity if the process of antibody labeling is nonoptimal. This is especially case for monoclonal antibodies in which all antibody molecules in a given preparation have almost the same affinity and so are most likely to be affected all together by improper labeling. This issue is less problematic for polyclonal antibodies in which antibodies with diverse physicochemical proper-

Needless to say, direct detection methods are the method of choice in such high-sensitive protein detection systems as flow cytometry. Although this system is the simplest and the most convenient method for detection of a given marker expression, it is not routinely employed in clinical and research applications due to

The need for more sensitive detection systems for antigens with low expression pattern was a provoking factor that prompted Coons et al. in 1941 to develop twostep detection methods [8]. This system employs an unlabeled primary antibody as the first layer and the secondary antibody, which is raised against the primary antibody and is labeled with different fluorophores or enzymes (**Figure 2**) [6, 9–11]. In indirect methods, primary antibodies retain full avidity because they remain unlabeled. Indeed, higher number of labels per molecule of primary antibody is achieved in indirect compared to direct detection methods. The later stems from the fact that at least two labeled secondary antibodies can bind to each primary antibody molecule. These factors result in increased reaction intensity and the higher sensitivity in indirect staining methods. Accordingly, indirect methods are able to detect fewer number of antigens with less primary antibody. Moreover, indirect methods are more practical than direct methods since the same secondary antibody can be applied for detection of different sets of primary antibodies if they have been raised in the same species [12]. Another benefit of indirect method in IF stainings is possibility to select secondary antibodies with fluorophores of different colors. For example, if the tissue shows strong endogenous red autofluorescence, the secondary antibody labeled with green

**Figure 1.** *Direct immunostaining method.*

### *Detection Systems in Immunohistochemistry DOI: http://dx.doi.org/10.5772/intechopen.82072*

*Immunohistochemistry - The Ageless Biotechnology*

tages of each method.

**2. Direct method**

tissue would be desirable.

*Direct immunostaining method.*

methods based on avidin-biotin interaction and polymer- and tyramide-based signal amplification are among IHC signal amplification methods that have greatly enhanced the sensitivity of IHC staining. However, when more sensitive methods are used, background signal tends to increase along with the target signal and so highly sensitive detection systems are not always desirable. Therefore, the optimal IHC method is planned as a compromise between sensitivity that allows proper and reliable visualization of a given molecular marker and at the same time avoiding background signals that impair staining index and specificity of the staining method. In an optimal IHC detection system, tissue type, level of expression of the marker of interest, localization of the marker, and cost are among important factors that should be taken into consideration. As a general rule of thumb, there is no a *bona fide* IHC detection method that is universally accepted. Although it does not rely on chemical reactions that take place in IHC, immunofluorescence (IF) staining follows almost the same rules as with IHC and so concerns on detection systems that are also applicable to IF staining methods. In this chapter, we will focus on detection methods in immunohistochemistry and immunofluorescence stainings and highlight in detail potential application, advantages, and disadvan-

Direct detection methods are known as a one-step process applying a primary antibody, which is directly labeled with reporter molecules, such as biotin, colloidal gold, fluorochromes, or enzymes [1, 2]. The conjugated antibody makes a direct contact with cognate antigen in histological or cytological preparations (**Figure 1**). Direct detection methods are widely used for detecting highly expressed antigens. Furthermore, when the use of the secondary antibodies causes nonspecific and unwanted reactions, owing to the histological nature of the tissue and/or host species of the primary antibody, direct detection could be the technique of choice. For instance, in case of mouse lymph node immunostaining, labeled primary mouse monoclonal antibodies are preferred because antimouse secondary antibodies are not only bound to mouse primary antibodies bound to the tissue antigens of interest but will also react with endogenous immunoglobulins vastly found in lymph nodes. This would lead to a strong nonspecific staining. Hence, direct detection methods using mouse primary antibodies conjugated to a fluorophore or enzyme would be a better option [3]. If this approach is not practically feasible, for example, due to the low expression level of the target antigen or technical problems in primary antibody labeling, indirect methods using primary antibodies from species other than that of target

One of the advantages of direct detection is that the incubation step with a secondary reagent is eliminated. Hence, this method is time saving and easy to

**4**

**Figure 1.**

perform. In addition, due to the wide range of fluorochromes that are commercially available, direct detection is vastly used in multicolor experimental designs [4, 5].

It is important to note that insufficient sensitivity to detect most of the antigens found in routinely processed tissues is one of the drawbacks of using direct detection method. Furthermore, each primary antibody needs to be individually conjugated with fluorophores or enzymes, which increases considerably the cost of the whole process. Another concern with direct staining methods is the possibility of functional impairment of the antibody affinity if the process of antibody labeling is nonoptimal. This is especially case for monoclonal antibodies in which all antibody molecules in a given preparation have almost the same affinity and so are most likely to be affected all together by improper labeling. This issue is less problematic for polyclonal antibodies in which antibodies with diverse physicochemical properties are produced against an antigen [5–7].

Needless to say, direct detection methods are the method of choice in such high-sensitive protein detection systems as flow cytometry. Although this system is the simplest and the most convenient method for detection of a given marker expression, it is not routinely employed in clinical and research applications due to the limitations mentioned above.
