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Published on: February 12, 2014
Double-label immunofluorescence with the laser scanning confocal microscope using cyanine dyes
1Department of Stomatology, University of California, San Francisco 94143, USA.
Insights
New immunofluorescence methods overcome autofluorescence in tissues. Using cyanine dyes (Cy3, Cy5) instead of fluorescein isothiocyanate (FITC) allows clear visualization of cellular structures in amphibian cardiac ganglia.
Area of Science:
- Immunofluorescence microscopy
- Cell biology
- Neuroscience
Background:
- Confocal microscopy enables simultaneous detection of multiple antigens.
- Traditional double-labeling uses fluorescein isothiocyanate (FITC) and Texas Red, excited by blue and yellow light, respectively.
- Autofluorescence in tissues, particularly at low wavelengths, can obscure signals when using FITC.
Purpose of the Study:
- To develop a superior immunofluorescence method for double-labeling studies in tissues with inherent autofluorescence.
- To overcome limitations of FITC-based detection in amphibian cardiac ganglion studies.
Main Methods:
- Utilized a krypton-argon ion laser scanning confocal microscope.
- Employed cyanine 3.18 (Cy3) and cyanine 5.18 (Cy5) conjugated secondary antibodies.
- Excited Cy3 and Cy5 with yellow (568 nm) and red (647 nm) laser lines, respectively, avoiding the problematic blue (488 nm) line.
Main Results:
- Cy3 and Cy5 antibodies emit in the orange/red and far-red spectrum, respectively.
- This method effectively avoids autofluorescence issues present with 488 nm excitation.
- Achieved image quality comparable to or better than FITC/Texas Red, even in autofluorescent tissues.
Conclusions:
- Cyanine dye-based immunofluorescence provides a robust solution for double-labeling in autofluorescent tissues.
- This technique enhances the analysis of cellular structures, such as receptor clusters and synaptic boutons, in challenging biological samples.
- The method offers improved visualization without the interference of low-wavelength autofluorescence.
Abstract:
The laser scanning confocal microscope, when used with the krypton-argon ion laser, is well suited for the simultaneous detection of pairs of antigens by immunofluorescence. Traditionally, double-label studies have utilized secondary antibodies conjugated to fluorescein isothiocyanate (FITC), excited by the 488-nm line (blue), and to tetramethyl rhodamine isothiocyanate or Texas Red, excited by the 568-nm line (yellow). However, the use of fluorophores excited by the 488 nm line produces unsatisfactory results when tissue contains low wavelength-excitable autofluorescence. In the amphibian cardiac ganglion, for example, autofluorescent granules within parasympathetic neurons obscure cell surface-derived signals and prevent one from analyzing the relative position of acetylcholine receptor clusters and synaptic boutons by double-label immunofluorescence. This problem has been solved by using cyanine 3.18 (Cy3)- and cyanine 5.18 (Cy5)-conjugated secondary antibodies, which are excited efficiently by the 568-nm (yellow) and the 647-nm (red) lines and which emit in the orange/red and in the far-red, respectively, and thus by avoiding the 488-nm line altogether. The resulting images are as good or better than those obtained with FITC and Texas Red, even without consideration of autofluorescence.
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