Target and signal amplification: approaches to increase the sensitivity of in situ hybridization

Abstract

In situ hybridization (ISH) has proven to be a very important molecular tool in research and diagnosis. However, its applicability can be limited by its restricted detection sensitivity. During the last few years, several strategies have been developed to improve the threshold levels for ISH detection by amplification of either target nucleic acid sequences prior to ISH or the detection signals after hybridization procedures. In this overview, we outline and analyze the principles, applications, and limitations of in situ polymerase chain reaction, in situ self-sustained sequence replication, primed in situ labeling (PRINS), and in situ transcription as examples of target amplification methods, and catalyzed reporter deposition using biotinylated tyramine as an approach to signal amplification in ISH. We also provide a detailed, 1-day protocol for non-radioactive oligonucleotide ISH including signal amplification, which is suitable for diagnostic purposes. Furthermore, future directions of ISH including combined strategies of target and signal amplification as well as automation are discussed.

PCR and in situ hybridization for the detection and localization of a new pathogen Francisella-like bacterium (FLB) in ornamental cichlids

“…No hybridization signal was obtained in the eye or the liver of the 4 fish tested. This might be due to the different infection stages or to the small number of bacterial particles that were present in these naturally infected fish because the threshold for detection is 20 copies of the gene of interest (Komminoth & Werner 1997). The DNA probe used for detection of FLB in tilapia has been used successfully to detect the agent causing visceral granulomas in ornamental fish.…”

“…Specific probes can be designed to locate organisms that cannot be cultured (Hayashi et al 1990, Komminoth & Werner 1997 or visualized by conventional methods, such as viruses (Lewis & Wells 1992), Chlamydiae (Campbell et al 1993) and Mycobacterium paratuberculosis (Hulten et al 2000). In addition to identifying the pathogens, ISH is an increasingly popular tool for locating pathogenic organisms in their target tissues (Gebhart et al 1994, Loy et al 1996, Gencay 1997, Gumus et al 1997, Jantos et al 1999, Chae et al 2002.…”

Immunohistochemical and hybridocytochemical study on ghrelin signalling in the rat seminiferous epithelium.

“…In case of ImmunoMax technique [39] after the incubation with anti-digoxygenin MAbs and a wash in PBS/Tween20, the biotinylated tyramine was applied at 1:50 dilution for 3 min at room temperature. The streptavidin-peroxidase complex was added for 30 min.…”

Expression of Cytokines (TNF-α, IL-1α, and IL-2) in Chronic Hepatitis C: Comparative Hybridocytochemical and Immunocytochemical Study in Children and Adult Patients

“…Two digoxygenin-labeled oligoprobes specific for human cytokine mRNA were used, including (a) human TNF-␣ probe cocktail (R&D Systems; Minneapolis, MN), (b) human IL-1 ␣ probe cocktail (R&D Systems), detected with sheep anti-digoxygenin monoclonal antibodies (MAbs) ń ń s´ń (Fab fragments), labeled with horseradish peroxidase (Roche). In the hybridocytochemical studies, the classical ones and those amplified by the ImmunoMax technique (Komminoth and Werner 1997), sequential sections of the tissue material were applied, which was pre-tested by ICC techniques. The ISH protocol of R&D Systems was employed with our own modification and ImmunoMax amplification of the signal.…”

“…Then the sections were treated with primary MAb at 1:20 dilution (anti-IL-1 ␣ ) and 1:100 dilution (anti-TNF-␣ and anti-IL-2) or ready-for-use dilutions (anti-CD68 and anti-human Von Willebrand Factor) overnight at 4C, then with the secondary biotinylated link of anti-mouse and anti-rabbit IgG and with the streptavidin-biotin-peroxidase complex (ABC) (DAKO). The studies followed the classical ABC technique (Hsu et al 1981), alone or associated with the ImmunoMax technique (Komminoth and Werner 1997). In our studies we did not use antigen retrieval but amplification of the final signal using biotinylated tyramine.…”