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Atto 465 Derivative Is a Nuclear Stain with Unique Excitation and Emission Spectra Useful for Multiplex Immunofluorescence Histochemistry

Atto 465 Derivative Is a Nuclear Stain with Unique Excitation and Emission Spectra Useful for... Multiplex immunofluorescence (mIF) is an effective technique for the maximal visualization of multiple target proteins in situ. This powerful tool is mainly limited by the spectral overlap of the currently available synthetic fluorescent dyes. The fluorescence excitation wavelengths ranging between 405 and 488 nm are rarely used in mIF imaging and serve as a logical additional slot for a fluorescent probe. In the present study, we demonstrate that the addition of 2,3,4,5,6-pentafluoroaniline to Atto 465 NHS ester, creating Atto 465-pentafluoroaniline (Atto 465-p), generates a bright nuclear stain in the violet-blue region of the visible spectrum. This allows the 405 nm excitation and emission, classically used for nuclear counterstains, to be used for the detection of another target protein. 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. (J Histochem Cytochem XX: XXX–XXX, XXXX) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Histochemistry & Cytochemistry SAGE

Atto 465 Derivative Is a Nuclear Stain with Unique Excitation and Emission Spectra Useful for Multiplex Immunofluorescence Histochemistry

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References (29)

Publisher
SAGE
Copyright
© The Author(s) 2022
ISSN
0022-1554
eISSN
1551-5044
DOI
10.1369/00221554211064942
Publisher site
See Article on Publisher Site

Abstract

Multiplex immunofluorescence (mIF) is an effective technique for the maximal visualization of multiple target proteins in situ. This powerful tool is mainly limited by the spectral overlap of the currently available synthetic fluorescent dyes. The fluorescence excitation wavelengths ranging between 405 and 488 nm are rarely used in mIF imaging and serve as a logical additional slot for a fluorescent probe. In the present study, we demonstrate that the addition of 2,3,4,5,6-pentafluoroaniline to Atto 465 NHS ester, creating Atto 465-pentafluoroaniline (Atto 465-p), generates a bright nuclear stain in the violet-blue region of the visible spectrum. This allows the 405 nm excitation and emission, classically used for nuclear counterstains, to be used for the detection of another target protein. 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. (J Histochem Cytochem XX: XXX–XXX, XXXX)

Journal

Journal of Histochemistry & Cytochemistry – SAGE

Published: Mar 1, 2022

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