PMID- 24637389
OWN - NLM
STAT- MEDLINE
DCOM- 20140721
LR  - 20240321
IS  - 1940-087X (Electronic)
IS  - 1940-087X (Linking)
IP  - 84
DP  - 2014 Feb 24
TI  - Fluorescence in situ hybridizations (FISH) for the localization of viruses and 
      endosymbiotic bacteria in plant and insect tissues.
PG  - e51030
LID - 10.3791/51030 [doi]
LID - 51030
AB  - Fluorescence in situ hybridization (FISH) is a name given to a variety of 
      techniques commonly used for visualizing gene transcripts in eukaryotic cells and 
      can be further modified to visualize other components in the cell such as 
      infection with viruses and bacteria. Spatial localization and visualization of 
      viruses and bacteria during the infection process is an essential step that 
      complements expression profiling experiments such as microarrays and RNAseq in 
      response to different stimuli. Understanding the spatiotemporal infections with 
      these agents complements biological experiments aimed at understanding their 
      interaction with cellular components. Several techniques for visualizing viruses 
      and bacteria such as reporter gene systems or immunohistochemical methods are 
      time-consuming, and some are limited to work with model organisms and involve 
      complex methodologies. FISH that targets RNA or DNA species in the cell is a 
      relatively easy and fast method for studying spatiotemporal localization of genes 
      and for diagnostic purposes. This method can be robust and relatively easy to 
      implement when the protocols employ short hybridizing, commercially-purchased 
      probes, which are not expensive. This is particularly robust when sample 
      preparation, fixation, hybridization, and microscopic visualization do not 
      involve complex steps. Here we describe a protocol for localization of bacteria 
      and viruses in insect and plant tissues. The method is based on simple 
      preparation, fixation, and hybridization of insect whole mounts and dissected 
      organs or hand-made plant sections, with 20 base pairs short DNA probes 
      conjugated to fluorescent dyes on their 5' or 3' ends. This protocol has been 
      successfully applied to a number of insect and plant tissues, and can be used to 
      analyze expression of mRNAs or other RNA or DNA species in the cell.
FAU - Kliot, Adi
AU  - Kliot A
AD  - Department of Entomology, Volcani Center; Institute of Plant Sciences and 
      Genetics in Agriculture, Robert H. Smith Faculty of Agriculture, Food and 
      Environment, Hebrew University of Jerusalem.
FAU - Kontsedalov, Svetlana
AU  - Kontsedalov S
AD  - Department of Entomology, Volcani Center.
FAU - Lebedev, Galina
AU  - Lebedev G
AD  - Department of Entomology, Volcani Center.
FAU - Brumin, Marina
AU  - Brumin M
AD  - Department of Entomology, Volcani Center.
FAU - Cathrin, Pakkianathan Britto
AU  - Cathrin PB
AD  - Department of Entomology, Volcani Center.
FAU - Marubayashi, Julio Massaharu
AU  - Marubayashi JM
AD  - Department of Entomology, Volcani Center.
FAU - Skaljac, Marisa
AU  - Skaljac M
AD  - Department of Entomology, Volcani Center; Department of Applied Sciences, 
      Institute for Adriatic Crops and Karst Reclamation.
FAU - Belausov, Eduard
AU  - Belausov E
AD  - The Institute of Plant Sciences, Volcani Center.
FAU - Czosnek, Henryk
AU  - Czosnek H
AD  - Institute of Plant Sciences and Genetics in Agriculture, Robert H. Smith Faculty 
      of Agriculture, Food and Environment, Hebrew University of Jerusalem.
FAU - Ghanim, Murad
AU  - Ghanim M
AD  - Department of Entomology, Volcani Center; ghanim@volcani.agri.gov.il.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Video-Audio Media
DEP - 20140224
PL  - United States
TA  - J Vis Exp
JT  - Journal of visualized experiments : JoVE
JID - 101313252
RN  - 0 (DNA, Bacterial)
RN  - 0 (DNA, Viral)
RN  - 0 (RNA, Bacterial)
RN  - 0 (RNA, Messenger)
RN  - 0 (RNA, Viral)
SB  - IM
MH  - Animals
MH  - Begomovirus/genetics/*isolation & purification
MH  - DNA, Bacterial/analysis
MH  - DNA, Viral/analysis
MH  - Female
MH  - Hemiptera/*microbiology/virology
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Solanum lycopersicum/microbiology/virology
MH  - Plant Diseases/*microbiology/virology
MH  - Plants/*microbiology/virology
MH  - RNA, Bacterial/analysis
MH  - RNA, Messenger/analysis
MH  - RNA, Viral/analysis
MH  - Symbiosis
PMC - PMC4130656
EDAT- 2014/03/19 06:00
MHDA- 2014/07/22 06:00
PMCR- 2016/02/24
CRDT- 2014/03/19 06:00
PHST- 2014/03/19 06:00 [entrez]
PHST- 2014/03/19 06:00 [pubmed]
PHST- 2014/07/22 06:00 [medline]
PHST- 2016/02/24 00:00 [pmc-release]
AID - 51030 [pii]
AID - 10.3791/51030 [doi]
PST - epublish
SO  - J Vis Exp. 2014 Feb 24;(84):e51030. doi: 10.3791/51030.