Comparison of anti-fading agents used in fluorescence microscopy: image analysis and laser confocal microscopy study.

Abstract

To ascertain the ability of commercial and home-made anti-fading media to reduce the decrease of fluorescein isothiocyanate (FITC) fluorescence, we studied the bleaching characteristics of FITC-stained Reh 6 cells mounted in buffered glycerol and in anti-fading media. We measured the intensity of fluorescence over time with a confocal laser scanning microscope and a standard epifluorescence microscope coupled to an image analysis system. Most of the anti-fading media effectively retard fading but each has drawbacks. Better results were obtained with media containing p-phenylenediamine (solutions in buffered glycerol, Vectashield, Fluorstop). However, Mowiol, Slowfade, n-propyl gallate (20 g/liter) were also effective in retarding fading. Most of them, except Mowiol, reduced fluorescence intensity. We concluded that the choice of anti-fading medium would depend on the desired results: a slower decay of fluorescence despite an initial quenching of fluorescence or a lower retardant effect with no decrease in initial fluorescence intensity. Moreover, the combination of Mowiol with another anti-fading medium may be a useful compromise when a strong retardant effect is required without marked quenching of the initial fluorescence.

Morphological and morphometrical assessment of spermathecae of Aedes aegypti females

“…Samples were then incubated for 30 min in fluorescent Phalloidin-FITC (Sigma-Aldrich) diluted 1:100 in 0.1% Triton X-100 and washed as before. Prior to analysis, samples were mounted with the anti-fading solution Mowiol (Longin et al 1993 and reference therein). Images were obtained using multiple confocal sections from a Zeiss laser confocal microscope 5100.…”

Anti-biotin Antibodies Offer Superior Organelle-specific Labeling of Mitochondria over Avidin or Streptavidin

“…Labeled cells were rinsed twice in PBS and fixed in methanol at Ϫ 20C for 5 min. The fixed coverslips were rinsed again in PBS and mounted in Moviol (Longin et al 1993).…”

Neural control of the velum in larvae of the gastropod,Ilyanassa obsoleta

“…Specimens were mounted in glycerol (3:1) in 0.1·mol·l -1 Tris buffer (pH·8.0) with 2% n-propyl gallate added to prevent fading (Longin et al, 1993) and then viewed with a Zeiss LSM 510 confocal microscope. Images were created by superimposing stacks of 10-80 images obtained through stepped sequences of focal planes at 0.10-0.80·mm intervals.…”