DiOC6(3) (3,3'-Dihexyloxacarbocyanine Iodide)

Since these dyes insert into lipid membranes, any disruption of the membranes leads to loss of the dye. This includes permeabilization with detergents like Triton X-100 or organic solvents like methanol. Permeabilization is necessary for intracellular antibody labeling, leading to loss of the dye. Instead, a reactive dye such as CFDA SE should be used to allow for covalent attachment to cellular components, thus providing for better retention upon fixation and permeabilization.

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The transport is fairly slow, around 6 mm/day in live tissue and slower in fixed tissue, so diffusion of lipophilic carbocyanine tracers from the point of their application to the terminus of a neuron can take several days to weeks The FAST DiO and DiI analogs (which have unsaturated alkyl tails) can improve transport rate by around 50%.

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Select the dye that is compatible with your available excitation source(s) and emission filter set/channels. The solid, paste and crystal forms can be applied directly to neurons in tissues. For labeling cells in culture or microinjection, the lipophilic dyes in solution or solid form can be used.

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Lipophilic cyanine dyes are preferred for this sort of assay, since they insert into cellular membranes and then, upon fusion, are shared by the fused cells as the membranes are shared. For example, one cell population can be labeled with DiI (orange-red) and another cell population can be labeled with DiO (green), and when the cells fuse, the combined color appears yellow (when imaged with a dual-bandpass filter set).

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