In situ Hybridization Histochemistry
Edythe D. London in Imaging Drug Action in the Brain, 2017
The digoxigenin method (recently marketed by Boehringer-Mannheim) utilizes similar principles as the biotin procedure. Digoxigenin is linked via a spacer arm to dUTP or UTP and incorporated into DNA or RNA via the labeling reactions described above. Detection occurs via incubation of hybrids with antidigoxigenin antibody conjugated to a reporter molecule (e.g., alkaline phosphatase) (Baldino and Lewis, 1989; Lewis et al., 1990). The sensitivity of this method seems superior to the biotin approach. Indeed, a method for labeling cRNA probes using digoxigenin has recently been developed and has been applied successfully toward detection of rare messages (Springer et al., 1991).
In Situ Hybridization
Attila Lorincz in Nucleic Acid Testing for Human Disease, 2016
Nonisotopic ISH methods are now commonly used.20–28 The first nonisotopic label was biotin, because of the high sensitivity of streptavidin detection systems. However, the widespread presence of endogenous biotin and the limited success of blocking methods stimulated the development of a range of other labels. Digoxigenin (DIG) is a derivative of the digoxin cardiac glycoside and can be used for probe labeling. DIG-labeled probes were more recently introduced and have become widely used, with higher sensitivity and less background staining than biotinylated probes.22–24
A Survey of Newer Gene Probing Techniques
Victor A. Bernstam in Pocket Guide to GENE LEVEL DIAGNOSTICS in Clinical Practice, 2019
The automated PCR/OLA system eliminates the need to measure DNA fragment sizes, the use of radioisotopes, as well as the requirement for high-quality DNA, particularly when polymorphic human STSs become available for automated forensic typing. The entire ELISA-based assay can be performed in microtiter plates, and no centrifugation or electrophoresis is needed. Digoxigenin-tagged oligonucleotides serve as reporter molecules in the ELISA of the duplexes captured via their biotinylated oligonucleotides.
Gestational folic acid content alters the development and function of hypothalamic food intake regulating neurons in Wistar rat offspring post-weaning
Published in Nutritional Neuroscience, 2020
Neil Victor Yang, Emanuela Pannia, Diptendu Chatterjee, Ruslan Kubant, Mandy Ho, Rola Hammoud, Zdenka Pausova, G. Harvey Anderson
The protocol for in situ hybridization staining was a modification of a protocol previously reported elsewhere.17 Briefly, DNA containing Npy and Pomc genes were isolated from hypothalamic brain tissue of Wistar rats using DNeasy Blood & Tissue Kit (Qiagen). Anti-sense Pomc and Npy digoxigenin-labeled cDNA probes were generated from the isolated DNA using the following primer sets:18Npy: (Forward) 5′-CTCCGCTCTGCGACACTAC-3′, (Reverse) 5′-AATCAGTGTCTCAGGGCT-3′, Pomc: (Forward) 5′-ACCTCACCACGGAGAGCA-3′, (Reverse) 5′-GCGAGAGAGTCGAGTTTGC-3′. DNA extracts were subject to DNA replication using KAPA2G Fast HotStart ReadyMix PCR Kit (KAPA Biosystems, MA, USA) according to manufacturer protocol. The digoxigenin-labeled Npy and Pomc probes were synthesized by Klenow enzyme and amplified by incorporating digoxigenin-labeled dUTP from the DIG DNA Labeling Mix (Sigma-Aldrich, MI, USA) during PCR.
The current and future applications of in situ hybridization technologies in anatomical pathology
Published in Expert Review of Molecular Diagnostics, 2022
Hoi Yi Leung, Martin Ho Yin Yeung, Wai Tung Leung, King Hin Wong, Wai Yan Tang, William Chi Shing Cho, Heong Ting Wong, Hin Fung Tsang, Yin Kwan Evelyn Wong, Xiao Meng Pei, Hennie Yuk Lin Cheng, Amanda Kit Ching Chan, Sze Chuen Cesar Wong
The century of the usage of the radioactive reporter was followed by the development of the non-radioactive species. Biotinylated labels successfully open another field of the reporting molecules involving enzymatic reaction together with the introduction of light microscopic (LM) and electron microscopic (EM) to ISH. Biotin-avidin is a demonstration of non-radioactive probe for ISH with the conjugation with alkaline phosphatase (AP) or horseradish peroxidase (HRP). The stability of the probe, outstanding resolution, and simpler experimental design makes them swift replacements of the radioisotope-labeling. In addition to biotin, digoxigenin is an alternative for the non-radioactive ISH detection. Digoxigenin-labeled probes can provide an even higher resolution and sensitivity than that of biotinylated labels due to its absence in mammalian tissues (17). The signals are visualized with the presence of anti-digoxigenin antibody and conjugation with AP or HRP. Nonetheless, the sensitivity and the ability of quantitation using biotin- or digoxigenin-labels can be further improved in subsequent years.
Generation of GM-CSF-producing antigen-presenting cells that induce a cytotoxic T cell-mediated antitumor response
Published in OncoImmunology, 2020
Hiroaki Mashima, Rong Zhang, Tsuyoshi Kobayashi, Yuichiro Hagiya, Hirotake Tsukamoto, Tianyi Liu, Tatsuaki Iwama, Masateru Yamamoto, Chiahsuan Lin, Ryusuke Nakatsuka, Yuta Mishima, Noriko Watanabe, Takashi Yamada, Satoru Senju, Shin Kaneko, Alimjan Idiris, Tetsuya Nakatsura, Hideki Ohdan, Yasushi Uemura
The cDNA fragment of murine Cd274 at cDNA positions 59–735 (GenBank accession number: NM_021893.3) was used for generation of sense or anti-sense RNA probes. Digoxigenin (DIG)-labeled RNA probes were prepared using DIG RNA Labeling Mix (Roche Diagnostics). Tissues were fixed with G-Fix (Genostaff), embedded in paraffin on CT-Pro20 (Genostaff) using G-Nox (Genostaff), as a less toxic solvent than xylene, and sectioned at 5 μm. In situ hybridization (ISH) was performed using the ISH Reagent kit (Genostaff) according to the manufacturer’s instructions. Paraffin-embedded sections were hybridized with the DIG-labeled RNA probes at 60°C for 16 h. After hybridization, sections were incubated with anti-DIG alkaline phosphate conjugate (Roche Diagnostics). The bound label was detected using alkaline phosphate color substrates nitro-blue tetrazolium chloride and 5-bromo-4-chloro-3ʹ-indolylphosphatase p-toluidine salt (Sigma-Aldrich). Sections were then counterstained with Kernechtrot Stain Solution (Muto Pure Chemicals, Tokyo, Japan) and mounted with G-Mount (Genostaff).
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