Atto 465 derivative is a nuclear stain with unique excitation and emission spectra for multiplex immunofluorescence histochemistry

The architecture of a biologic response is inextricably linked with the tissue architecture of the target site. Multiplex immunofluorescence (mIF) is an effective technique for the maximal visualization of multiple target proteins in situ. This powerful tool is limited by the spectral overlap separation of the currently available synthetic fluorescent dyes. The fluorescence excitation wavelengths ranging between 405nm and 488nm are rarely used in mIF imaging and serve as a logical additional slot for a fluorescent probe. In the present study, we demonstrate that Atto 465-pentafluoroaniline (Atto 465-p), a derivative of the fluorescent dye Atto 465, can serve as a nuclear stain in the violet-blue region of the visible spectrum. This opens the 405nm channel, traditionally used for nuclear stain, for detection of another experimental target. This increases the flexibility of the mIF panel and, with appropriate staining and microscopy, enables the quantitative analysis of at least six targets in one tissue section.