What is the secret to getting good fluorescent separation in multi-colour staining?
There are a few things that will improve signal to noise ratio. • Pick the best flurochrome for you antibodies. Do not pick a dimly fluorescent fluorochrome for an antibody that will have few receptors binding sites or where the cell of interest will be a rare event Use fluorochromes such as PE or APC. A good reference is the Fluorochrome Intensity Chart by eBioscience. • Avoid antibody under saturation and over saturation by titrating antibodies. Under saturation will result in weak signals and poor separation between negative and positive populations. Too much antibody (over saturation) will result in unbound antibody, causing unnecessary noise. Do not rely on manufacturers suggest dilution.
