FM 4-64 is a lipophilic styryl dye and a vital stain: it fluoresces only in living cells, so cells cannot be fixed then stained nor stained then fixed. You must stain and observe living cells. FM 4-64 does not permeate cell membranes but, instead, intercalates into the plasma membrane is then taken into the cells by endocytosis.

Protocol: FM 4-64 Labeling of Yeast Vacuole Membranes Protocol