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Saturation Analysis: Radioimmunoassay and Other Ligand Assays
Published in Joseph Chamberlain, The Analysis of Drugs in Biological Fluids, 2018
On-line immunochemical detection in liquid chromatography has been described using fluorescein-labeled antibodies.1227 The eluale is mixed with polyclonal antidigoxigenin Fab labeled with fluorescein, next a column of immobilized digoxin removes free antibodies, and then measurement is by fluorescence. Sensitivities of 200 fmol digoxin and 50 fmol digoxigenin were reported.
In situ Hybridization Histochemistry
Published in Edythe D. London, Imaging Drug Action in the Brain, 2017
Martin K.-H. Schofer, James P. Herman, Stanley J. Watson
We have recently applied digoxigenin-labeled cRNA probes for detection of neuropeptide mRNAs in brain and endocrine tissues. Figure 2 shows a comparison of provasopressin mRNA localization in the hypothalamic paraventricular nucleus, using [35S]- and digoxigenin-labeled probes. Both methods show robust labeling over magnocellular neurons in this region; note, however, the vastly superior resolution afforded by the digoxigenin-labeled, immunochemically detected probe (with reaction product localized within the cytoplasmic compartment of discrete cells). Detection times vary from 2 to 24 h for the digoxigenin method, whereas emulsion-dipped auto-radiographs must be exposed from several days to weeks before a robust signal can be generated, illustrating the great time savings offered by the nonradioactive method. However, the digoxigenin cRNA method is not yet as sensitive as standard autoradiographic detection; in our hands, there is still difficulty in detection of rare messages (e.g., enkephalin mRNA in the pituitary gland) using nonradioactive labeling protocols.
In Situ Hybridization
Published in Attila Lorincz, 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
MicroRNA-122-5p ameliorates tubular injury in diabetic nephropathy via FIH-1/HIF-1α pathway
Published in Renal Failure, 2022
Li Cheng, Xinying Qiu, Liyu He, Li Liu
Fluorescence in situ hybridization (FISH) was performed according to the manufacturer's instructions. Briefly, kidneys were harvested from control and STZ-treated mice to prepare 4-micron paraffin section. The sections were treated with 20 μg/ml proteinase K for permeabilization, and then incubated with pre-hybridization solution at 78 °C for 1 h. Remove pre-hybridization solution and add digoxigenin-labeled mmu-miR-122-5p LNA probe over night at 37 °C. At the second day, after wash, bovine serum albumin (BSA) was added for blocking. Then the anti-digoxigenin-HRP was used at 37 °C for 1 h. CY3-TSA and DAPI assay were used to indicate the positive areas and cell nucleus respectively. The images were acquired from a fluorescence microscope and the representative figures were exhibited.
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.
Ebselen oxide attenuates mechlorethamine dermatotoxicity in the mouse ear vesicant model
Published in Drug and Chemical Toxicology, 2020
Hemanta C. Rao Tumu, Benedette J. Cuffari, Maria A. Pino, Jerzy Palus, Magdalena Piętka-Ottlik, Blase Billack
During the apoptotic process, DNA strand breaks occur as part of DNA fragmentation. In the TUNEL assay, the TdT enzyme catalyzes the addition of digoxigenin labeled and unlabeled nucleotides to the free 3′-OH termini of these DNA strand breaks. The incorporated nucleotides then form an oligomer composed of both digoxigenin nucleotides and unlabeled nucleotides. The digoxigenin-labeled nucleotides are then allowed to bind with fluorescein-conjugated anti-digoxigenin antibody. The apoptotic nuclei can be viewed by fluorescence microscopy using standard fluorescein excitation and emission filters. The assay was performed as described by the manufacturer (EMD Millipore Corporation, Billerica, MA; Cat # S7110). A Leica DMIL inverted microscope equipped with SLF-130-LX ScopeLED fluorescence system and Luminera Infinity 3 mono camera with Infinity Capture and Infinity Analyze 6.5.4 was used for obtaining fluorescence microscopy images.