Analysis of antifading reagents for fluorescence microscopy

Abstract

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...

Developmental selection within the angiosperm style: Using gamete DNA to visualize interspecific pollen competition

“…Embedded flowers were serially sectioned into 5-m thick ribbons and mounted on microscope slides. Slides were flooded with a solution of 0.25 g͞ml of 4Ј,6-diamidino-2-phenylindole (DAPI) and 0.1 mg͞ml p-phenylenediamine (added to reduce fading) (13) in 0.05 M Tris (pH 7.2) for 1 hr at room temperature in a light-free environment. Microspectrofluorometric measurements of relative DNA levels of DAPI-stained nuclei were performed within 2 hr (for detailed methods, see ref.…”

Double Immunolabeling of Neuropeptides in the Human Hypothalamus as Analyzed by Confocal Laser Scanning Fluorescence Microscopy

“…If the intensity of the laser excitation is kept at a moderate level, fading of the fluorophores FITC and Cy3 is minimal and background autofluorescence is negligible, while the fluorescence signal is generally sufficient for qualitative and quantitative analysis ( Figures 1B-1F). For more details about antifading agents, the reader is referred to Florijn et al (1995).…”

Double Fertilization in Gnetum gnemon: The Relationship between the Cell Cycle and Sexual Reproduction.

“…Later, ovules were dehydrated through an ethanol series, embedded in glycol methacrylate, and serially sectioned as previously described. The resulting slides were flooded with asolution of 0.5 vg/mL DAPl and 0.1 mglmL pphenylenediamine (added to reduce fading) (Florijn et al, 1995) in phosphate-buffered saline, pH 7.2, for 15 min at room temperature in a light-free environment. Coverslips were then mounted, and the slides were placed in a dark, humid environment for an additional 60 min.…”