I stained my cells with a lipophilic cyanine dye, like DiI, but the signal was lost when I tried to follow up with antibody labeling. Why?
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.