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Journal of Histochemistry and Cytochemistry, Vol. 48, 437-444, March 2000, Copyright © 2000, The Histochemical Society, Inc.

TECHNICAL NOTE

Direct Eye Visualization of Cy5 Fluorescence for Immunocytochemistry and In Situ Hybridization

Gian-Luca Ferria, Jorma Isolab, Peter Bergerc, and Gabriele Girod
a Neuro-&-Endocrine Research, Department of Cytomorphology, University of Cagliari and Oasi IRCCS, Troina, Italy
b Institute of Medical Technology, Tampere University and University Hospital, Finland
c Institute for Biomedical Aging Research, Austrian Academy of Sciences, Innsbruck, Austria
d FRAE Institute, CNR, Bologna, Italy

Correspondence to: Gian-Luca Ferri, Dept. of Cytomorphology, University of Cagliari at Monserrato, I-09042 Monserrato (Cagliari), Italy.

Cyanine 5.18 (or Cy5) is a fluorochrome emitting in the long-red/far-red range, usually regarded as unsuitable for direct observation by the human eye. We describe here the optimization of a direct visualization approach to Cy5 labeling, based on a standard fluorescence microscope with mercury light excitation and applicable to both immunocytochemistry and fluorescent in situ hybridization. Crucial factors were (a) an excitation path in the microscope not absorbing light in the orange-red range, up to 640 nm, (b) a 588–640-nm excitation filter range, distinctly below the excitation optimum for Cy5, (c) a 650–700-nm emission filter range, transmitting the low-wavelength portion of Cy5 emission, and (d) high-efficiency filter set components allowing a narrow gap between excitation and emission ranges without visible cross-talk of excitation light in the emission path. (J Histochem Cytochem 48:437–444, 2000)

Key Words: Cy5, fluorescence, immunohistochemistry, immunofluorescence, FISH, multiple staining, fluorescence filter, fluorochrome


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