Comparison of anti-fading agents used in fluorescence microscopy: image analysis and laser confocal microscopy study.
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.
Longin
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A
Laboratory of Analytical Cytology, University Claude Bernard, Lyon, France.
Souchier
C
C
Ffrench
M
M
Bryon
P A
PA
D000818:Animals; D003470:Culture Media / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate; D005453:Fluorescence; D005990:Glycerol; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008853:Microscopy / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D010655:Phenylenediamines / Q000592:standards*; D010879:Piperazines / Q000592:standards*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000473:pathology; D011435:Propyl Gallate / Q000592:standards*; D011837:Radiation-Protective Agents / Q000592:standards*; D014407:Tumor Cells, Cultured
D003470:Culture Media; D010655:Phenylenediamines; D010879:Piperazines; D011837:Radiation-Protective Agents; D011435:Propyl Gallate; D016650:Fluorescein-5-isothiocyanate; D005990:Glycerol; C029728:4-phenylenediamine; C007306:triethylenediamine