**3. Indirect method**

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 2.** *Indirect immunostaining method.*

fluorophore could be a right choice [3]. Previously mentioned advantages of indirect detection systems eventually led to its widespread applications in research and clinical settings.

Despite advantages mentioned above, indirect immunostaining methods suffer from some shortcomings. First, additional controls and blocking steps are inevitable when using secondary antibodies. Indeed, there is possibility of nonspecific staining that happens when the secondary antibody interacts with unwanted tissue targets. If nonspecific staining is noticed, blocking reagents have to be used to treat the tissue sections that could be time-consuming and cause additional costs to IHC experiment [6, 13]. The blocking agent should contain nonimmune antibody fraction from the same species in which secondary antibody has been produced. This results in competitive blocking of the nonspecific binding sites for secondary antibody in the target tissue by the unlabeled antibodies from the same species. The addition of further layers beyond their use in the two-step indirect method for increasing the sensitivity of detection can be problematic as addition of every new species of antibody considerably increases the risk of nonspecific interactions and background staining [14]. Desire for more sensitive detection systems triggered researchers to develop next-generation detection systems, especially for those antigenic markers, which are not expressed in physiological condition, and any level of their upregulation would be interpreted as a pathological condition.
