Resistance of Dictyostelium membranes to saponin permeabilization is dependent on osmotic conditions. Cells incubated in various conditions were fixed, permeabilized with saponin, stained with H161 antibody (green), then permeabilized with methanol and labeled again with H161 antibody (red) as described in the Legend to figure 1. A) Cells grown in HL5 medium. Arrowheads indicate the few compartments that were only stained after methanol permeabilization. B) Cells exposed to SB starvation buffer. C) Cells exposed to an isotonic starvation buffer (SB+100 mM sorbitol) were efficiently permeabilized with saponin suggesting that osmotic conditions, rather than starvation, modulate resistance of Dictyostelium membranes to saponin permeabilization. All the pictures presented in this figure were from the same experiment, and taken consecutively with identical microscope settings, allowing direct comparison of labeling intensities.