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

Abstract

Objective: Synthetic fluorescent dyes that are conjugated to antibodies are useful tools toprobe molecules. Based on dye chemical structures, their photobleaching and photostabilityindices are quite diverse. It is generally believed that among different fluorescent dyes,Alexa Fluor family has greater photostability than traditional dyes like fluorescein isothiocyanate(FITC) and Cy5. Alexa Fluor 568 is a member of Alexa Fluor family presumed tohave superior photostability and photobleahing profiles than FITC.Materials and Methods: In this experimental study, we conjugated Alexa Fluor 568 andFITC dyes to a mouse anti-human nestin monoclonal antibody (ANM) to acquire their photobleachingprofiles and photostability indices. Then, the fluorophore/antibody ratios werecalculated using a spectrophotometer. The photobleaching profiles and photostability indicesof conjugated antibodies were subsequently studied by immunocytochemistry (ICC).Samples were continuously illuminated and digital images acquired under a fluorescentmicroscope. Data were processed by ImageJ software.Results: Alexa Fluor 568 has a brighter fluorescence and higher photostability thanFITC.Conclusion: Alexa Fluor 568 is a capable dye to use in photostaining techniques and ithas a longer photostability when compared to FITC.

Keywords

• Fluorescein Isothiocyanate
• Alexa Fluor 568
• Photostability
• Photobleaching