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Conjugates of AMCA absorb light maximally at 350 nm and fluoresce maximally at 450 nm (Table 1 and Figure 2). For fluorescence microscopy, AMCA can be excit-ed with a mercury lamp and observed using a UV filter set. Since blue fluorescence is not well detected by the human eye, AMCA-conjugated secondary antibodies should be used with the most abundant antigens in multiple-labeling experiments. Ways of improving the visibility of AMCA include dark adapting the eyes, using fluorite instead of glass objectives, avoiding mounting media that absorb UV light (such as plastic-based media), and capturing photographic images. AMCA also fades as rapidly as fluorescein; and therefore, it should be used with an anti-fading agent.
For flow cytometry, AMCA can be excited with a mercury lamp, or with a water-cooled argon ion laser which emits some lines in the UV region of the spectrum.
Table 1: Approximate* peak wavelengths of absorption and emission for different fluorophore-conjugated, affinity-purified
antibodies.
| Fluorophore |
Absorption Peak (nm) |
Emission Peak (nm) |
| Aminomethylcoumarin, AMCA |
350 |
450 |
| Cyanine, Cy2 |
492 |
510 |
| Fluorescein, FITC |
492 |
520 |
| Indocarbocyanine, Cy3 |
550 |
570 |
| Tetramethyl Rhodamine, TRITC |
550 |
570 |
| Rhodamine Red-X, RRX |
570 |
590 |
| Texas Red, TR |
596 |
620 |
| Indodicarbocyanine, Cy5 |
650 |
670 |
*Only approximate values are given for purposes of comparing one fluorophore with another.
Actual values may vary
depending on the spectrofluorometer used in each laboratory.
Figure 2. Excitation and emission spectra of different fluorophore-conjugated, affinity-purified antibodies. This figure illustrates only the relative shape and position of each fluorophore in the peak region of its excitation and emission following conjugation to antibodies. Quantitative comparisons should not be made since peak heights have been normalised. All spectra were obtained with a M-Series spectrofluorometer system from Photon Technology International, Inc. |
AMCA has been used mostly for multiple labeling since there is minimal fluorescence overlap with fluorescein and little or no overlap with longer wavelength-emitting fluorophores. Applications for multiple labeling with this probe include both immunofluorescence microscopy and flow cytometry. AMCA is not suggested for single labeling because of its relatively weak signal.
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