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

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.