EVALUATION OF 5 GREEN FLUORESCENCE-EMITTING STREPTAVIDIN-CONJUGATED FLUOROCHROMES FOR USE IN IMMUNOFLUORESCENCE MICROSCOPY

Fluorescein isothiocyanate (FITC) is largely used in immunofluorescenc e methods. We propose to analyse the quality of some recent fluorochro mes using image analysis. Fluorochromes tested include FITC and dichlo rotriazinylaminofluorescein (DTAF), dipyrrometheneboron difluoride (BO DIPY), Rhodol Green and cyanine 2. RAMOS cells were immunolabelled aga inst the proliferating cell nuclear antigen (PCNA) revealed by the bio tin-streptavidin technique. Slides were mounted in anhydrous glycerol or in buffered glycerol (pH 7.0 or pH 8.5). No antifading medium was a dded. Cell fluorescence emission intensity and bleaching characteristi cs were measured. Rhodol Green exhibited the highest fluorescence inte nsity and the best photobleaching resistance, Although BODIPY also res isted well during the photobleaching assay, its fluorescence intensity was weak. FITC, DTAF and cyanine 2 showed intermediate fluorescence i ntensity and a fast decay of fluorescence. Among the green emitting fl uorochromes tested, Rhodol Green appeared to be the best.