Figure 1

Assessing permeabilization by detergents in Dictyostelium cells. A) In order to test the permeabilization efficiency of saponin, fixed cells were permeabilized with saponin and incubated with H161 antibodies coupled to Alexa-488 (green). The cells were then further permeabilized with methanol at -20°C, and incubated with the H161 antibody coupled to Alexa-647 (red). Labeled cells were visualized in a LSM510 Zeiss confocal microscope. In cells grown in HL5 medium, the variable amounts on red and green labeling in various compartments indicated significant variations in the efficiency of saponin permeabilization. A few compartments were only stained after methanol permeabilization (arrowheads). B) Cells were treated as described in A, but saponin was replaced with Triton X-100. Triton permeabilized efficiently cellular membranes. C) In cells incubated for 30 minutes in starvation buffer (SB), virtually no intracellular staining was seen after saponin permeabilization, suggesting that membranes were highly resistant to saponin permeabilization. D) Cells exposed to SB were efficiently permeabilized with Triton X-100. 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 in various conditions.