Comparison of Photostability and Photobleaching Properties of FITC- and Dylight488- Conjugated Herceptin

ABSTRACT Nowadays, fluorescent dyes have become significant tools in immunostaining experiments. Different fluorescent dyes have various physicochemical traits. Some of these traits, for instance photobleaching and photostability, are applicable calibers in such experiments as immunocytochemistry (ICC), immunohistochemistry (IHC), and flow cytometry (FC). In this study, photobleaching, photostability, and mean fluorescence intensity (MFI) of FITC (fluorescein isothiocyanate)- and Dylight488- conjugated Herceptin, the humanized anti Her2 monoclonal antibody drug, were examined. After conjugation of FITC and Dylight488 to Herceptin, their degree of labeling (DOL) was calculated by a spectrophotometer. Photobleaching, photostability, and MFI of conjugates were subsequently investigated by ICC and FC on BT-474 cells. Dylight488 labeled Herceptin revealed higher fluorescent lifetime and photostability than FITC conjugated Herceptin.