U1snRNA, U3snRNA, 28 S ribosomal RNA, poly(A) RNA and a specific messenger RNA were visualized in living cells with microinjected fluorochrome-labeled 2' O-Methyl oligoribonucleotides (2' OMe RNA). Antisense 2' OMe RNA probes showed fast hybridization kinetics, whereas conventional oligodeoxyribonucleotide (DNA) probes did not. The nuclear distributions of the signals in living cells were similar to those found in fixed cells, indicating specific hybridization. Cytoplasmic ribosomal RNA, poly(A) RNA and mRNA could hardly be visualized, mainly due to a rapid entrapment of the injected probes in the nucleus. The performance of linear probes was compared with that of molecular beacons, which due to their structure should theoretically fluoresce only upon hybridization. No improvements were achieved however with the molecular beacons used in this study, suggesting opening of the beacons by mechanisms other than hybridization. The results show that linear 2' OMe RNA probes are well suited for RNA detection in living cells, and that these probes can be applied for dynamic studies of highly abundant nuclear RNA. Furthermore, it proved feasible to combine RNA detection with that of green fluorescent protein-labeled proteins in living cells. This was applied to show co-localization of RNA with proteins and should enable RNA-protein interaction studies.

The utility of p-phenylenediamine, 1,4-di-azobicyclo-(2.2.2.)-octane, and the commercial products Citifluor, Slowfade, and Vectashield, antifading agents frequently used as mounting media for fluorescence in situ hybridization, was investigated. Fading curves for bound fluorochromes were recorded with digital microscopy, and relative fluorescence intensities of fluorochromes in solution were measured with an aperture defined measurement system. The three commonly used fluorochromes, fluorescein, tetramethyl rhodamine, and coumarin, were studied. Vectashield offered the best antifading properties for all three fluorochromes, although their relative fluorescence intensity was slightly less in Vectashield in comparison with other antifading agents. In Vectashield, fluorescein, tetramethyl rhodamine, and coumarin showed half-life times of 96,330, and 106 s, respectively, whereas in 90% glycerol in PBS (pH SS), these half-life time values were 9, 7, and 25 s, respectively. Vectashield is particularly recommended as a mounting medium for quantitative digital imaging microscopy and for multicolor applications, where it is easy to have errors due to differences in fading rates of the fluorochromes. Key terms: Fluorescence microscopy, antifading agents, DABCO, p-phenylenediamine, Vectashield, fluorescence in situ hybridization (FISH) Immunofluorescence (IF) and fluorescence in situ hybridization (FISH) techniques are important tools in both hndamental and applied biomedical research. At present a variety o f fluorochromes can be coupled to proteins or nucleic acids (7J2). Among the most commonly applied fluorochromes are fluorescein. rhodamine, and coumarin derivatives. They provide the three primary colours of the visible part of the electromagnetic spectrum and ample possibilities for multicolour studies, especially in multicolour FISH with combinatorial and ratio-labelling approaches ( 55.1 2,13,17,2 2 ).The minimal amount of target-bound fluorochrome that can be detected microscopically is dependent on the microscope setup as well as o n the absorption coefficient and quantum efficiency of the fluorophore. Additionally, the utility of a fluorochrome depends on its fading properties because of the extended periods of excitation time that are necessary for visual evaluation and photography or for image acquisition with cooled (slow-scan) charge coupled device (CCD) cameras.Fading o f fluorochromes upon excitation is a photochemical process Light-induced damage to the fluorochrome is prominent in the presence of oxygen ( 6 ) . Also, nonoxygen-mediated radical generation has been indicated as a source of photochemical fluorochrome destruction (9). Consequently, the fading characteristics can be influenced by adding compounds to the mounting media, such as antioxidants and radical scavengers, which interfere with the photochemical reaction in such a way that the excited fluorochrome will not be (irreversibly) damaged. p-Phenylenediamine (I'd) and 1,4-diaza-bicyclo-( 2,2.2)-octane (DABCO), both antioxydants, arc two we...

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SummaryOver a decade ago the idea of endothelial cell seeding was introduced in an attempt to improve the function of small caliber vascular prostheses. Although endothelial cell seeding is currently being applied clinically, several questions regarding the functional properties of the seeded endothelial cells remain. Evaluation of functional properties of endothelial cells on various types of vascular prostheses can be performed partly in vitro, but it is hampered by the fact that commonly used methods to quantify endothelial cells do not adequately apply to these cells on prosthetic materials.An accurate quantification method is described that is rapidly and easily applicable to endothelial cells attached to vascular prostheses. The method can also be used to quantify endothelial cells attached to culture dishes or microcarriers. Colorless, non-fluorescing, fluorescein-di-acetate was used, which was taken up by the attached endothelial cells, and which was then intracellularly converted to yellow fluorescein, emitting green fluorescence. Subsequently, triton-X-100 was appli-cated to release fluorescein and levels of fluorescence were measured with the automated aperture-defined microvolume (ADM) method, using an inverted fluorescence microscope to which a photometer was connected. The measured level of fluorescence is linearly related to endothelial cell numbers attached to prostheses. The accuracy and the reproducibility of cell countings are high.