Studies of the Ecophysiology of Single Cells in Microbial Communities by (Quantitative) Microautoradiography and Fluorescence In Situ Hybridization (MAR-FISH)

2015 ◽  
pp. 115-130 ◽  
Author(s):  
Marta Nierychlo ◽  
Jeppe Lund Nielsen ◽  
Per Halkjær Nielsen
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Microbial Communities ◽  
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.

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