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Detection of transcripts via fluorescence in-situ hybridization and confocal microscopy in whole mounts of roots of Arabidopsis thaliana

Serge Bauwens (UGent) , Els Verhoeyen (UGent) , Janice de Almeida Engler (UGent) , Marc Van Montagu (UGent) and Gilbert Engler (UGent)
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Abstract
We report a modification of a previously published procedure (de Almeida Engler et al., 1994) aimed at the fluorescent detection of RNA-RNA hybrids. Transcripts of ARAth;Cyc1;1 were located in whole mounts of roots of Arabidopsis thaliana after in-situ hybridization with digoxigenin- and biotin-labeled antisense probes. Digoxigenin- and biotin-labeled hybrids were detected by FISH with an AP-conjugated antibody against digoxigenin or AP-conjugated streptavidin for detection of biotin, with the UV-excitable dye ELF(TM) as a substrate. Alternatively, biotin-labeled hybrids were detected with HRP-conjugated streptavidin, with fluorescein tyramine as a substrate. Images were recorded via confocal microscopy. We report on the different fixation and reaction conditions needed for obtaining the highest signal-to-noise ratio, as well as on the requirements for mounting media, filter sets, excitation wavelengths, and recording modes used for recording confocal data sets.
Keywords
Arabidopsis thaliana, DEPOSITION, transcripts, confocal microscopy, fluorescence insitu hybridization, cyclin 1

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Chicago
Bauwens, Serge, Els Verhoeyen, Janice de Almeida Engler, Marc Van Montagu, and Gilbert Engler. 1997. “Detection of Transcripts via Fluorescence In-situ Hybridization and Confocal Microscopy in Whole Mounts of Roots of Arabidopsis Thaliana.” Plant Molecular Biology Reporter 15 (1): 22–37.
APA
Bauwens, Serge, Verhoeyen, E., de Almeida Engler, J., Van Montagu, M., & Engler, G. (1997). Detection of transcripts via fluorescence in-situ hybridization and confocal microscopy in whole mounts of roots of Arabidopsis thaliana. PLANT MOLECULAR BIOLOGY REPORTER, 15(1), 22–37.
Vancouver
1.
Bauwens S, Verhoeyen E, de Almeida Engler J, Van Montagu M, Engler G. Detection of transcripts via fluorescence in-situ hybridization and confocal microscopy in whole mounts of roots of Arabidopsis thaliana. PLANT MOLECULAR BIOLOGY REPORTER. 1997;15(1):22–37.
MLA
Bauwens, Serge, Els Verhoeyen, Janice de Almeida Engler, et al. “Detection of Transcripts via Fluorescence In-situ Hybridization and Confocal Microscopy in Whole Mounts of Roots of Arabidopsis Thaliana.” PLANT MOLECULAR BIOLOGY REPORTER 15.1 (1997): 22–37. Print.
@article{182813,
  abstract     = {We report a modification of a previously published procedure (de Almeida Engler et al., 1994) aimed at the fluorescent detection of RNA-RNA hybrids. Transcripts of ARAth;Cyc1;1 were located in whole mounts of roots of Arabidopsis thaliana after in-situ hybridization with digoxigenin- and biotin-labeled antisense probes. Digoxigenin- and biotin-labeled hybrids were detected by FISH with an AP-conjugated antibody against digoxigenin or AP-conjugated streptavidin for detection of biotin, with the UV-excitable dye ELF(TM) as a substrate. Alternatively, biotin-labeled hybrids were detected with HRP-conjugated streptavidin, with fluorescein tyramine as a substrate. Images were recorded via confocal microscopy. We report on the different fixation and reaction conditions needed for obtaining the highest signal-to-noise ratio, as well as on the requirements for mounting media, filter sets, excitation wavelengths, and recording modes used for recording confocal data sets.},
  author       = {Bauwens, Serge and Verhoeyen, Els and de Almeida Engler, Janice and Van Montagu, Marc and Engler, Gilbert},
  issn         = {0735-9640},
  journal      = {PLANT MOLECULAR BIOLOGY REPORTER},
  keyword      = {Arabidopsis thaliana,DEPOSITION,transcripts,confocal microscopy,fluorescence insitu hybridization,cyclin 1},
  language     = {eng},
  number       = {1},
  pages        = {22--37},
  title        = {Detection of transcripts via fluorescence in-situ hybridization and confocal microscopy in whole mounts of roots of Arabidopsis thaliana},
  url          = {http://dx.doi.org/10.1007/BF02772110},
  volume       = {15},
  year         = {1997},
}

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