Comparison of the Photobleaching and Photostability Traits of Alexa Fluor 568- and Fluorescein Isothiocyanate- conjugated Antibody

Objective: synthetic fluorescent dyes that are conjugated to antibodies are useful tools to probe molecules. based on dye chemical structures, their photobleaching and photostabil- ity indices are quite diverse. it is generally believed that among different fluorescent dyes, alexa fluor family has greater photostability than traditional dyes like fluorescein isothio- cyanate (fitc) and cy5. alexa fluor 568 is a member of alexa fluor family presumed to have superior photostability and photobleahing profiles than fitc. materials and methods: in this experimental study, we conjugated alexa fluor 568 and fitc dyes to a mouse anti-human nestin monoclonal antibody (anm) to acquire their pho- tobleaching profiles and photostability indices. then, the fluorophore/antibody ratios were calculated using a spectrophotometer. the photobleaching profiles and photostability indi- ces of conjugated antibodies were subsequently studied by immunocytochemistry (icc). samples were continuously illuminated and digital images acquired under a fluorescent microscope. data were processed by imagej software. results: alexa fluor 568 has a brighter fluorescence and higher photostability than fitc. conclusion: alexa fluor 568 is a capable dye to use in photostaining techniques and it has a longer photostability when compared to fitc.