Publisher Summary This chapter describes the agar-overlay method that overcomes two major problems faced by conventional immunofluorescence. These are (1) drastic shrinkage and/or deformation of the cells caused by formaldehyde fixation, and (2) insufficient resolution because of superimposition of the fluorescence because of the round or cylindrical shape of Dictyostelium cells. These problems are overcome by flattening the living cells with an overlay of a thin agarose sheet while conducting instantaneous fixation with cold absolute methanol. This procedure results in cells that are 1–2 pm thick and 20–30 pm wide and aids orientation of the filamentous structures in parallel to the focal plane. Using high-titer monoclonal anti-bodies, specific distributions of actin, myosin, and tubulin are described in Dictyostelium. It is shown that the myosin filaments transiently change their distribution responding to a chemotactic stimulus in a dynamic manner. “Agar-overlay” immunofluorescence seems to be generally useful for small and round vertebrate nonmuscle cells and those of lower organisms. This technique can provide valuable information on the dynamic features and the detailed organization of cytoskeletal elements that, otherwise, cannot be visualized with sufficient resolution.