O-106 6-Color simultaneous detection of 5 prenatal aneuploidies in single cells by fluorescence in situ hybridization (FISH)

1997 ◽  
Vol 68 ◽  
pp. S53
Author(s):  
L.E Morrison ◽  
M.S Legator
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Single Cells ◽  
Simultaneous Detection

scholarly journals Combined immunocytochemistry and fluorescence in situ hybridization for simultaneous tricolor detection of cell cycle, genomic, and phenotypic parameters of tumor cells.

1994 ◽  
Vol 42 (7) ◽  
pp. 961-966 ◽  
Author(s):  
E J Speel ◽  
J Herbergs ◽  
F C Ramaekers ◽  
A H Hopman
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Tumor Cells ◽  
Simultaneous Detection ◽  
Dna Probes ◽  
Accurate Localization ◽  
Fast Red ◽  
Pre Treatment ◽  
High Fluorescence

We describe the development and application of a sensitive high-resolution fluorescence alkaline phosphatase (APase)-Fast Red immunocytochemical (ICC) staining method in combination with fluorescence in situ hybridization (ISH) and bromodeoxyuridine (BrdU) detection. The high fluorescence intensity, accurate localization, and advantageous slow-fading properties make the APase-Fast Red reaction a valuable tool for detection of antigens or specific DNA probes in biological cell preparations. Since the enzyme precipitate proved to be resistant to enzymatic pre-treatment steps and stable during the entire ISH procedure, APase-Fast Red immunostaining could be combined with subsequent visualization of DNA target sequences by fluorescence ISH. The lung cancer cell lines NCI-H82 and EPLC 65 were used as a model system for simultaneous detection of cell proteins, such as the neural cell adhesion molecule (N-CAM), cytokeratin filaments, lamin or the Ki67 antigen (Ki67-Ag), and centromere-specific DNA probes for human chromosomes 1, 7, or 17. In addition, the combined ICC/ISH procedure could be extended with the immunodetection of BrdU incorporated by tumor cells in S-phase. As a consequence, a combined ICC/ISH/BrdU detection procedure is now available that enables analysis of relatively complex tumor populations on the basis of different ICC and genetic markers as well as proliferative activity.

scholarly journals Fluorescence in Situ Hybridization (FISH) for Detecting Anaplastic Lymphoma Kinase (ALK) Rearrangement in Lung Cancer: Clinically Relevant Technical Aspects

2019 ◽  
Vol 20 (16) ◽  
pp. 3939 ◽  
Author(s):  
Zhenya Tang ◽  
Lu Wang ◽  
Guilin Tang ◽  
L. Jeffrey Medeiros
Keyword(s):  
Lung Cancer ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Simultaneous Detection ◽  
The United States ◽  
Driver Mutations ◽  
Alk Rearrangement ◽  
Technical Aspects ◽  
Anaplastic Lymphoma

In 2011, the Vysis Break Apart ALK fluorescence in situ hybridization (FISH) assay was approved by the United States Food and Drug Administration as a companion diagnostic for detecting ALK rearrangement in lung cancer patients who may benefit from treatment of tyrosine kinase inhibitor therapy. This assay is the current “gold standard”. According to updated ALK testing guidelines from the College of American Pathologists, the International Association for the Study of Lung Cancer and the Association for Molecular Pathology published in 2018, ALK immunohistochemistry is formally an alternative to ALK FISH, and simultaneous detection of multiple hot spots, including, at least, ALK, ROS1, RET, MET, ERBB2, BRAF and KRAS genes is also recommended while performing next generation sequencing (NGS)-based testing. Therefore, ALK status in a specimen can be tested by different methods and platforms, even in the same institution or laboratory. In this review, we discuss several clinically relevant technical aspects of ALK FISH, including pros and cons of the unique two-step (50- to 100-cell) analysis approach employed in the Vysis Break Apart ALK FISH assay, including: the preset cutoff value of ≥15% for a positive result; technical aspects and biology of discordant results obtained by different methods; and incidental findings, such as ALK copy number gain or amplification and co-existent driver mutations. These issues have practical implications for ALK testing in the clinical laboratory following the updated guidelines.

scholarly journals FISH-Flow, a protocol for the concurrent detection of mRNA and protein in single cells using fluorescence in situ hybridization and flow cytometry

2017 ◽  
Vol 12 (6) ◽  
pp. 1245-1260 ◽  
Author(s):  
Riccardo Arrigucci ◽  
Yuri Bushkin ◽  
Felix Radford ◽  
Karim Lakehal ◽  
Pooja Vir ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Single Cells

scholarly journals Simultaneous Fluorescence In Situ Hybridization of mRNA and rRNA in Environmental Bacteria

2004 ◽  
Vol 70 (9) ◽  
pp. 5426-5433 ◽  
Author(s):  
Annelie Pernthaler ◽  
Rudolf Amann
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Single Cells ◽  
False Negative ◽  
Proteinase K ◽  
Hybridization Temperature ◽  
Fish Samples ◽  
Different Types ◽  
Pure Cultures

ABSTRACT We developed for Bacteria in environmental samples a sensitive and reliable mRNA fluorescence in situ hybridization (FISH) protocol that allows for simultaneous cell identification by rRNA FISH. Samples were carbethoxylated with diethylpyrocarbonate to inactivate intracellular RNases and pretreated with lysozyme and/or proteinase K at different concentrations. Optimizing the permeabilization of each type of sample proved to be a critical step in avoiding false-negative or false-positive results. The quality of probes as well as a stringent hybridization temperature were determined with expression clones. To increase the sensitivity of mRNA FISH, long ribonucleotide probes were labeled at a high density with cis-platinum-linked digoxigenin (DIG). The hybrid was immunocytochemically detected with an anti-DIG antibody labeled with horseradish peroxidase (HRP). Subsequently, the hybridization signal was amplified by catalyzed reporter deposition with fluorochrome-labeled tyramides. p-Iodophenylboronic acid and high concentrations of NaCl substantially enhanced the deposition of tyramides and thus increased the sensitivity of our approach. After inactivation of the antibody-delivered HRP, rRNA FISH was performed by following routine protocols. To show the broad applicability of our approach, mRNA of a key enzyme of aerobic methane oxidation, particulate methane monooxygenase (subunit A), was hybridized with different types of samples: pure cultures, symbionts of a hydrothermal vent bivalve, and even sediment, one of the most difficult sample types with which to perform successful FISH. By simultaneous mRNA FISH and rRNA FISH, single cells are identified and shown to express a particular gene. Our protocol is transferable to many different types of samples with the need for only minor modifications of fixation and permeabilization procedures.

scholarly journals A Fluorescence in Situ Hybridization Method To Quantify mRNA Translation by Visualizing Ribosome–mRNA Interactions in Single Cells

2017 ◽  
Vol 3 (5) ◽  
pp. 425-433 ◽  
Author(s):  
Kelly S. Burke ◽  
Katie A. Antilla ◽  
David A. Tirrell
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Single Cells ◽  
Mrna Translation ◽  
Hybridization Method
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