AMCA conjugates absorb light maximally at around 350 nm and fluoresce maximally at around 450 nm (Table 2 and Figure 13). For fluorescence microscopy, AMCA can be excited 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 only 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 with blue-sensitive film or CCD cameras. AMCA fades rapidly in conventional epifluorescence and confocal microscopy, and therefore it should be used with mounting media containing an anti-fading agent, such as n-propyl gallate.


Table 2. Approximate peak wavelengths of excitation and emission for JIR conventional and DyLight fluorophores conjugated to affinity-purified antibodies. 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.

Excitation	Emission