| Technical Information on Probes Conjugated to Affinity-Purified Antibodies and Other Proteins: Cyanine Dyes (Cy2, Cy3, and Cy5) |
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Cy2 conjugates can be excited at 492 nm and fluoresce (510 nm) in the green region of the visible spectrum (Table 1 and Figure 2) like FITC (520 nm) but are more photostable, less sensitive to pH changes, and more fluorescent in organic mounting media than FITC. Because of these properties, Cy2 can be visualized for a longer time in an epifluorescence microscope, and appears brighter than FITC. The same filters used for FITC are appropriate for use with Cy2.
Caution: It is important to avoid the use of mounting media containing p-phenylenediamine, since this anti-fading agent reacts with Cy2, resulting in weak and diffused fluorescence after storage of stained slides. Cy3 and Cy5 are brighter, more photostable, and give less background than most other fluorophores. Cy3 conjugates can be excited maximally at 550 nm, with peak emission at 570 nm (Table 1 and Figure 2). For fluorescence microscopy, they can be visualized with traditional tetramethyl rhodamine (TRITC) filter sets since the excitation and emission spectra (Figure 2) are nearly identical to those of TRITC. Table 1: Approximate* peak wavelengths of absorption and emission for different fluorophore-conjugated, affinity-purified antibodies.
*Approximate values are given for purposes of comparing one fluorophore with another. Actual values may vary slightly depending on the spectrofluorometer used in each laboratory.
Cy3 can be excited to about 50% of maximum with an argon laser (514 nm or 528 nm lines), or to about 75% of maximum with a helium/neon laser (543 nm line) or mercury lamp (546 nm line). Cy3 has been used with fluorescein for double labeling. However, the use of a narrow band-pass emission filter for fluorescein is recommended to minimize Cy3 fluorescence in the FITC filter set. Cy3 can also be paired with Cy5 for multiple labeling when using a confocal microscope.
Cy5 conjugates are excited maximally at 650 nm and fluoresce maximally at 670 nm (Table 1 and Figure 2). They can be excited to about 98% of maximum with a krypton/argon laser (647 nm line) or to about 63% of maximum with a helium/neon laser (633 nm line). Cy5 can be used with a variety of other fluorophores for multiple labeling due to a wide separation of its emission from that of shorter wavelength-emitting fluorophores. A significant advantage of using Cy5 over otherfluorophores is the low autofluorescence of biological specimens in this region of the spectrum. However, because of its emission maximum at 670 nm, Cy5 cannot be seen well by eye, and it cannot be excited optimally with a mercury lamp. Therefore, it is not recommended for use with conventional epifluorescence microscopes. It is most commonly visualized with a confocal microscope equipped with an appropriate laser for excitation and a far-red detector. Although anti-fading agents usually are not required when visualizing cyanine dye conjugates in an epifluorescence microscope, anti-fading agents should be added to aqueous mounting media for confocal laser scanning microscopy. For speci-mens labeled with Cy2, avoid the use of mounting media containing p-phenylenediamine, since this anti-fading agent reacts with Cy2, resulting in weak and diffused fluorescence after storage of stained slides. Other anti-fading agents, such as n-propyl gallate, may be used for mounting cyanine dye stained sections in aqueous media. Organic based mounting media, such as DPX or methyl salicylate, may also be used with cyanine dyes.
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