Genomic in situ hybridization (GISH) was conducted as previously described with minor modifications (25 (link)). Maize genomic DNA was labeled with digoxigenin–11-dUTP or biotin–11-dUTP (Roche) via nick translation. The digoxigenin- and biotin-labeled probes were incubated with anti-digoxigenin antibody conjugated with rhodamine (Roche) and anti-avidin antibody conjugated with fluorescein isothiocyanate (Vector Laboratories), respectively. Chromosomes were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) in an anti-fade solution. Fluorescence in situ hybridization (FISH) analyses on root tip chromosomes were performed according to procedures as previously described (26 (link),27 (link)). Slides were also counterstained with 4′, 6-diamino-phenylindole (Vector Laboratories). An epifluorescence microscope (Olympus BX61) attached to a CCD camera (QImaging; RETGA-SRV FAST 1394) was used to capture FISH/GISH images. Image-Pro Plus 6.0 software (Media Cybernetics) was used to analyze and adjust all digital images.
Anti digoxigenin antibody conjugated with rhodamine
Manufactured by Roche
The anti-digoxigenin antibody conjugated with rhodamine is a laboratory reagent used for detection and quantification purposes. It binds to the digoxigenin molecule, which is commonly used as a label in various molecular biology techniques. The rhodamine fluorescent dye attached to the antibody allows for the visualization and localization of the target molecules.
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2 protocols using anti digoxigenin antibody conjugated with rhodamine
1
Genomic in situ Hybridization of Maize
2
Fluorescent In Situ Hybridization of Pseudis tocantins
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Fragments of PcP190 and 5S rDNA sequences obtained from Pseudis tocantins as described above were labeled with digoxigenin-12-dUTP (Roche) using the PCR Dig Probe Synthesis Kit (Roche). Labeled DNA was co-precipitated with sonicated salmon sperm DNA (100 ng/μL) using 3M sodium acetate (1/10 volume) and ethanol. The pellet was washed in 70% ethanol and resuspended in hybridization buffer (50% formamide, 2x SSC and 10% dextran sulfate). The hybridization protocol was performed according to Viegas-Péquignot [37 ]. Digoxigenin-labeled probes were detected using an anti-digoxigenin anti-body conjugated with rhodamine (Roche), following the manufacturer´s instructions. Chromosomes were stained with DAPI (0.5 μg/mL). For the analysis of the PcP-1a probes, a control experiment was done, which consisted in their hybridization with metaphase chromosomes of an exemplar of Physalaemus aff. cuvieri (ZUEC 17897). Images were captured on an Olympus Bx60 fluorescence microscope and edited using Adobe Photoshop CS3 or/and Image ProPlus 4.0 (Media Cybernetics).
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