Procedure for whole mount fluorescence in situ hybridization of interphase nuclei on Arabidopsis thaliana |
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| Authors: | Serge Bauwens Katerina Katsanis Marc Van Montagu Patrick Van Oostveldt and Gilbert Engler |
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| Institution: | Laboratorium voor Genetica, Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium; Laboratorium voor Biochemie en Moleculaire Cytologie, Universiteit Gent, B-9000 Gent, Belgium; Laboratoire Associéde l'Institut National de la Recherche Agronomique (France), Universiteit Gent, B-9000 Gent, Belgium |
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| Abstract: | A procedure for whole mount fluorescence in situ hybridization (FISH) on plant tissue is reported. The technique was demonstrated on seedlings and flowers of Arabidopsis thaliana L. with rDNA as a probe, labelled, both for direct and indirect detection. It was found that fixation in 1% formaldehyde yielded the best results with respect to morphology and hybridization efficiency. The combination of whole mount FISH and confocal scanning laser microscopy allowed the nuclear localization of the rDNA loci in all tissues of both seedlings and flowers. Direct labelling yielded the best signal-to-noise ratio, especially in the apical zones of the seedlings. The technique was further illustrated on seedlings of A. thaliana in double labelling experiments with rDNA and a tandemly repeated, 500 bp sequence of A. thaliana. Although nuclei in all tissues in the seedling exhibited both signals, hybridization efficiency for both signals was reduced in the dense, apical zones as compared with single labelling experiments with rDNA. |
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