Fluorescence In Situ Hybridization on Single Cells

2007 ◽  
pp. 19-30 ◽  
Author(s):  
Paul N. Scriven ◽  
Caroline Mackie Ogilvie
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Single Cells

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.

l-DNA-tagged fluorescence in situ hybridization for highly sensitive imaging of RNAs in single cells

2020 ◽  
Vol 18 (40) ◽  
pp. 8084-8088
Author(s):  
Motoyuki Ogata ◽  
Gosuke Hayashi ◽  
Anri Ichiu ◽  
Akimitsu Okamoto
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Single Cells ◽  
Rna Detection ◽  
Pcr Product ◽  
Highly Sensitive

l-DNA tagged FISH (LT-FISH), including two-step hybridization processes with a l–d chimera oligonucleotide and a fluorescence-labeled PCR product tethering a l-DNA tag, has realized sensitive RNA detection in fixed cells.

scholarly journals Fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) for accurate detection and counting of RNA copies in single cells

2017 ◽  
Vol 46 (2) ◽  
pp. e7-e7 ◽  
Author(s):  
Yi Cui ◽  
Dehong Hu ◽  
Lye Meng Markillie ◽  
William B Chrisler ◽  
Matthew J Gaffrey ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Single Cells ◽  
Accurate Detection ◽  
Detection And Counting

Abstract 2895: Phylogenetic models of tumor progression from fluorescence in situ hybridization (FISH) data on many single cells of a solid tumor.

2013 ◽  
Author(s):  
Salim A. Chowdhury ◽  
Alejandro A. Schäffer ◽  
Stanley E. Shackney ◽  
Darawalee Wangsa ◽  
Kerstin Heselmeyer-Haddad ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Tumor Progression ◽  
Solid Tumor ◽  
Single Cells ◽  
Phylogenetic Models

scholarly journals Linking Microbial Phylogeny to Metabolic Activity at the Single-Cell Level by Using Enhanced Element Labeling-Catalyzed Reporter Deposition Fluorescence In Situ Hybridization (EL-FISH) and NanoSIMS

2008 ◽  
Vol 74 (10) ◽  
pp. 3143-3150 ◽  
Author(s):  
Sebastian Behrens ◽  
Tina Lösekann ◽  
Jennifer Pett-Ridge ◽  
Peter K. Weber ◽  
Wing-On Ng ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Metabolic Activity ◽  
Secondary Ion Mass Spectrometry ◽  
Single Cells ◽  
Target Cells ◽  
Filamentous Cyanobacterium ◽  
Novel Approach ◽  
Catalyzed Reporter Deposition

ABSTRACT To examine phylogenetic identity and metabolic activity of individual cells in complex microbial communities, we developed a method which combines rRNA-based in situ hybridization with stable isotope imaging based on nanometer-scale secondary-ion mass spectrometry (NanoSIMS). Fluorine or bromine atoms were introduced into cells via 16S rRNA-targeted probes, which enabled phylogenetic identification of individual cells by NanoSIMS imaging. To overcome the natural fluorine and bromine backgrounds, we modified the current catalyzed reporter deposition fluorescence in situ hybridization (FISH) technique by using halogen-containing fluorescently labeled tyramides as substrates for the enzymatic tyramide deposition. Thereby, we obtained an enhanced element labeling of microbial cells by FISH (EL-FISH). The relative cellular abundance of fluorine or bromine after EL-FISH exceeded natural background concentrations by up to 180-fold and allowed us to distinguish target from non-target cells in NanoSIMS fluorine or bromine images. The method was optimized on single cells of axenic Escherichia coli and Vibrio cholerae cultures. EL-FISH/NanoSIMS was then applied to study interrelationships in a dual-species consortium consisting of a filamentous cyanobacterium and a heterotrophic alphaproteobacterium. We also evaluated the method on complex microbial aggregates obtained from human oral biofilms. In both samples, we found evidence for metabolic interactions by visualizing the fate of substrates labeled with 13C-carbon and 15N-nitrogen, while individual cells were identified simultaneously by halogen labeling via EL-FISH. Our novel approach will facilitate further studies of the ecophysiology of known and uncultured microorganisms in complex environments and communities.

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