Fluor x dctp

The DNA probes used for FISH were pTr18S (GenBank accession number AF071069), a 1.8 kb fragment from Trifoliumrepens Linnaeus, 1753 containing almost the entire 18S rDNA sequence representing the 35S rDNA and pTr5S (GenBank accession number AF072692), a 596 bp DNA fragment encoding the T.repens 5S rRNA. 35S and 5S rDNA probes were directly labelled with fluorochromes Fluor-X-dCTP and Cy-3-dCTP (GE Healthcare, NZ), respectively by nick translation according to manufacturer’s specifications. Double target FISH using the above DNA probes, post-hybridisation washing and counterstaining of somatic chromosomes with DAPI were carried out as described earlier (Ansari et al. 1999 (link)). Chromosome preparations were mounted in Vectashield (Vector Laboratories). Fluorescence images were acquired using a Zeiss monochrome MRm CCD camera on a Nikon epifluorescence microscope Microphot-SA and were processed with an ISIS FISH Imaging System (MetaSystems, Germany). At least five good quality early to late metaphase cells from each plant were used for analysing hybridization signals.

Fluorescence in situ hybridization (FISH) was performed according to Pimpinelli et al. [72 ].
Squashed preparations of polytene chromosomes from salivary glands dissected from third instar larvae of D. virilis were denatured and hybridized with Cy3-dCTP or FluorX-dCTP (GE Healthcare) labeled probes. CG41265 and CG17665 were simultaneously localized using mixed probes. Polytene chromosomes were stained with DAPI, 4’, 6’-diamidine-2’-phenylindole-dihydrochloride. Chromosome preparations were analyzed using a computer controlled Nikon E1000 epifluorescence microscope equipped with a cooled CCD camera (Coolsnap). Digital images were obtained using an Olympus epifluorescence microscope equipped with a cooled CCD camera. Gray scale images, obtained by separately recording Cy3, FluorX and DAPI fluorescence with specific filters, were pseudo colored and merged for the final image using Adobe Photoshop. Labelled sites were identified on the basis of published polytene maps [35 (link)].