• Choose the brightest set of fluorochromes for your instrument.
• Minimize the spectral overlap through choosing the right fluorescent labels.
• Combine the brightest label with the protein which has the lowest level of expression, and vice versa.
• When using secondary antibodies - All the primary antibodies have been raised in different species to ensure cross labeling via the secondary antibodies is not encountered. The secondary antibodies recognize one of the species exclusively.
• Sample autofluorescence is sometimes a problem and must be taken in to consideration when choosing your antibody's fluorochrome conjugated. Check the specificity of the signal by fluorescence microscopy. Avoiding those channels when choosing fluorochromes or making sure that the signal from your fluorochromes is significantly higher than your autofluorescence.
For multi-color analysis there is a preference for scientists to use secondary antibodies conjugated to fluorescent labels. Due to their novel electronic configurations, fluorochromes have a unique and characteristic spectra for absorption and emission - a single dye is excited at a particular wavelength and emits a photon at another wavelength. Alternatively, a tandem dye consists of a donor and acceptor fluorochrome molecules being placed in close proximity, allowing for energy transfer between the two. The tandem dye is excited at the excitation wavelength of the acceptor molecule and emits a photon at the emission wavelength of the donor molecule.