**8. Rolling circle amplification**

*Immunohistochemistry - The Ageless Biotechnology*

treatment of tissues to quench endogenous peroxidase or endogenous avidin-biotin activities (EABA) is usually necessary [105, 107–109]. Although TSA/CSA detection methods have resulted in satisfactory results in terms of significantly increased sensitivity in IHC and *in situ* hybridization (ISH), they are not widely employed in diagnostic pathology. The reasons include: additional steps that make the method more time-consuming, nonspecific background staining, and that optimal AR treatment with existing methods may achieve equivalent results and that secondgeneration polymer-based methods are simpler and equally sensitive [14, 110, 111].

In an attempt to reduce the problems associated with endogenous biotin in conventional tyramide signal amplification, a biotin-free system, fluorescyl-tyramide amplification system (FT-CSA or CSAII), was introduced. Rather than biotinyltyramide, this system uses fluorescyl-tyramide and does not contain avidin/biotin reagents avoiding the problem associated with endogenous biotin. In this method, addition of primary antibody is followed by a peroxidase-labeled secondary antibody. Peroxidase enzyme is responsible to catalyze the transformation and deposition of fluorescyl-tyramide in the tissue section. When the reaction terminates, it could be inspected by fluorescence microscopy. The produced signals could even be converted to a colorimetric reaction by using peroxidase-conjugated antifluorescein

This method is highly sensitive enabling researchers to detect and localize antigens with low expression level and to use primary antibodies with very low affinities [105, 106]. Alternative reporter includes dinitrophenol, which also results in marked reduction of background from endogenous biotin. Absence of nonspecific staining

In the latest improvement of the biotin-free CSA method, fluorescein is conserved in the substrate, while the tyramine is substituted with ferulic acid, which is a much better peroxidase substrate and increases signal-to-noise ratio. In this system, the incubation time in each step can be significantly reduced, making it

antibody and a diaminobenzidine-hydrogen peroxide substrate.

is due to no endogenous tissue distribution of dinitrophenol [14].

possible to stain a tissue in less than 1 h [112].

**14**

**7.2 Biotin-free TSA/CSA**

*Tyramide-based immunostaining method.*

**Figure 7.**

Rolling circle amplification (RCA) reaction was first developed for the purpose of nucleic acid detection [106], but it was then adapted for amplification of signals from antibodies bound to antigens [113–118]. RCA is an enzymatic process in which a short DNA or RNA primer is amplified using a circular DNA template and special DNA or RNA polymerases to form a long single-stranded DNA or RNA [119, 120]. The end product of RCA is a long continuous sequence of DNA containing several tandem repeats complementary to the circular template. Unlike PCR, RCA could be performed at a constant temperature (room temperature to 37°C). A RCA reaction contains five different components: (i) a short DNA or RNA primer, (ii) a polymerase enzyme (e.g., Phi29 DNA polymerase for DNA, and T7 RNA polymerase for RNA), (iii) a suitable buffer compatible with polymerase enzyme, (iv) a circular DNA template, and (v) deoxy nucleotide triphosphates (dNTPs) [121].

RCA reaction has three different steps: (1) the circular DNA template with typically ~15–200 nt in length is synthesized through the intramolecular ligation of phosphate and hydroxyl end groups of a linear probe with the use of the target DNA or RNA as a ligation template [121–123], (2) the polymerase enzyme continuously adds dNTPs to a circular template-annealed primer to form a long ssDNA with tens to hundreds of tandem repeats, and (3) the RCA end products could be detected and even monitored by different signal readout methods (**Figure 8**) [121]. Different methods are available to visualize and also analyze the RCA process including (a) labeling the RCA products directly during the amplification process by using

#### **Figure 8.**

*Rolling circle amplification immunostaining method. (1) Immunoconjugate bound to target antigen. (2) RCA primer hybridized to circle template (3) Synthesis of new DNA strand by DNA polymerase (4) Detection of amplified DNA by enzyme-labeled probe at the site of bound antibody.*

fluorescent dyes-conjugated dNTPs; (b) detecting the RCA product with hybridization of fluorophore-tethered complementary strands; quantum dots or gold nanoparticles can be attached to RCA products via a complimentary strand to visualize RCA product; (c) using molecular beacon for fluorescent detection of RCA products; (d) using DNA binding dyes such as SYBR green; (e) using biotinylated decorator and streptavidin-HRP conjugate or by DNA-peptide nucleic acid (PNA) intercalating dye for colorimetric detection of RCA product; and (f) using luciferase to generate light for bioluminescence detection of RCA products [121].

One of the important advantages of RCA is that circular templates can be customized so that the signals of a single binding event are amplified in an exponential manner (e.g., multiprimed RCA) [124–126]. In this approach, signal amplification more than 109 -fold is feasible, while a linear mode of RCA has a capacity to amplify signals to nearly 105 -fold [127]. RCA reactions could be accomplished on a solid surface and also in a solution environment. In solid-phase RCA, reaction is conducted on a solid surface such as glass, microwell plates, microbead or nanobead particles, paper strips, or microfluidic devices. This system gives researchers an advantage of high-throughput analysis and potential for easy detection of target from complex sample matrices [121].

RCA is appeared to be a powerful method in immunoassays. The combination of RCA method with ELISA is found to grant more sensitivity and decrease the lower detection limit. In this regard, there is an approach called immuno-RCA in which, a RCA primer-conjugated antibody is applied on a target antigen that has been coated on a solid surface followed by a RCA reaction. The first immuno-RCA test was introduced by Schweitzer B et al. (2000) on a glass slide for IgE quantification [128]. From that time, solid-phase RCA has become popular as signal amplification method in antibody microarray analysis of multiplexed proteins [129–132]. A sandwich immuno-RCA has been adapted to detect the target with high sensitivity. In this technique, the target antigen in biological media is first captured on a solid surface using coated antibody. In the next step, a RCA primer-conjugated secondary antibody is applied to conduct the RCA reaction [133–135].

Konry et al. [136], by combining the capacity of RCA reaction to detect a singlemolecule and microfluidic technology, demonstrated the feasibility of identification of specific protein markers on tumor cell surfaces in miniaturized nanoliter reaction droplets. This approach of signal amplification in a microfluidic format could improve the applicability of existing methods by reducing consumption of sample and reagent and increasing the specificity and sensitivity for various applications such as early diagnosis of cancer [136]. Specific immunocytochemical and immunohistochemial identification of a wide range of intracellular molecules (prostate-specific antigen and vimentin) and cell surface antigens (epithelial membrane antigen, CD3 and CD20) in a variety of tissues (tonsil and breast) and cell lines (U266, Jurkat) has successfully been accomplished using RCA-mediated signal amplification. Indeed, immuno-RCA was reported to give more uniform staining pattern compared to the conventional methods [129]. *In situ* proximity ligation assays (*in situ* PLA) are an important adaptation of the RCA method in which primary antibodies against two distinct antigens are applied. Having two antigens in close proximity to each other, RCA reaction will occur and the proximity of two distinct antigens can be visualized [137].

It has been shown that attachment of a RCA primer to primary or secondary antibody does not impair affinity or avidity of the conjugate. Nonetheless, RCA reaction adds 60–90 min to the conventional IHC protocol. Although RCA is able to generate amplification of DNA up to 109 -fold, immuno-RCA in LSAB-based IHC applications is able to increase the signal to only about fourfold [129]. Simultaneous evaluation of TSA and RCA detection techniques by Warford et al. revealed that both methods are capable to produce results with a high signal-to-noise ratio. However, they found TSA detection system to be more sensitive than the RCA method [14].

**17**

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

avidin-biotin activity (EABA) [141].

of cancer [143, 144].

**Acknowledgements**

cal staining during experimental researches.

amplification [138].

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

The sensitivity of an IHC staining is a function of detection method for signal

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

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

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

The authors dedicate this book chapter to all mice, which generously made substantial contribution for improving authors' knowledge of immunohistochemi-

procedure and help surgeon to obtain wider surgical margins if needed.

*Immunohistochemistry - The Ageless Biotechnology*

than 109

to nearly 105

fluorescent dyes-conjugated dNTPs; (b) detecting the RCA product with hybridization of fluorophore-tethered complementary strands; quantum dots or gold nanoparticles can be attached to RCA products via a complimentary strand to visualize RCA product; (c) using molecular beacon for fluorescent detection of RCA products; (d) using DNA binding dyes such as SYBR green; (e) using biotinylated decorator and streptavidin-HRP conjugate or by DNA-peptide nucleic acid (PNA) intercalating dye for colorimetric detection of RCA product; and (f) using luciferase to generate light for bioluminescence detection of RCA products [121]. One of the important advantages of RCA is that circular templates can be customized so that the signals of a single binding event are amplified in an exponential manner (e.g., multiprimed RCA) [124–126]. In this approach, signal amplification more


also in a solution environment. In solid-phase RCA, reaction is conducted on a solid surface such as glass, microwell plates, microbead or nanobead particles, paper strips, or microfluidic devices. This system gives researchers an advantage of high-throughput analysis and potential for easy detection of target from complex sample matrices [121]. RCA is appeared to be a powerful method in immunoassays. The combination of RCA method with ELISA is found to grant more sensitivity and decrease the lower detection limit. In this regard, there is an approach called immuno-RCA in which, a RCA primer-conjugated antibody is applied on a target antigen that has been coated on a solid surface followed by a RCA reaction. The first immuno-RCA test was introduced by Schweitzer B et al. (2000) on a glass slide for IgE quantification [128]. From that time, solid-phase RCA has become popular as signal amplification method in antibody microarray analysis of multiplexed proteins [129–132]. A sandwich immuno-RCA has been adapted to detect the target with high sensitivity. In this technique, the target antigen in biological media is first captured on a solid surface using coated antibody. In the next step, a RCA primer-conjugated secondary

Konry et al. [136], by combining the capacity of RCA reaction to detect a singlemolecule and microfluidic technology, demonstrated the feasibility of identification of specific protein markers on tumor cell surfaces in miniaturized nanoliter reaction droplets. This approach of signal amplification in a microfluidic format could improve the applicability of existing methods by reducing consumption of sample and reagent and increasing the specificity and sensitivity for various applications such as early diagnosis of cancer [136]. Specific immunocytochemical and immunohistochemial identification of a wide range of intracellular molecules (prostate-specific antigen and vimentin) and cell surface antigens (epithelial membrane antigen, CD3 and CD20) in a variety of tissues (tonsil and breast) and cell lines (U266, Jurkat) has successfully been accomplished using RCA-mediated signal amplification. Indeed, immuno-RCA was reported to give more uniform staining pattern compared to the conventional methods [129]. *In situ* proximity ligation assays (*in situ* PLA) are an important adaptation of the RCA method in which primary antibodies against two distinct antigens are applied. Having two antigens in close proximity to each other, RCA reaction will

antibody is applied to conduct the RCA reaction [133–135].

occur and the proximity of two distinct antigens can be visualized [137].

TSA detection system to be more sensitive than the RCA method [14].

It has been shown that attachment of a RCA primer to primary or secondary antibody does not impair affinity or avidity of the conjugate. Nonetheless, RCA reaction adds 60–90 min to the conventional IHC protocol. Although RCA is able to generate

is able to increase the signal to only about fourfold [129]. Simultaneous evaluation of TSA and RCA detection techniques by Warford et al. revealed that both methods are capable to produce results with a high signal-to-noise ratio. However, they found



**16**

amplification of DNA up to 109
