Fig. 2. M-C₆-NBD-PC is not trafficked to the vacuole via the endocytic pathway. Strains were grown to early log phase in SDC at 23°C. After a 30 minute incubation at 37°C, cells were labeled for 1 hour with 50 µM of M-C₆-NBD-PC-containing vesicles as described in the Materials and Methods. The cells were then washed three times with ice-cold SCNaN₃ and harvested prior to imaging by fluorescence microscopy. The cells shown were labeled at the restrictive temperature, 37°C. When labeled at the permissive temperature, 23°C, both strains also exhibited vacuolar labeling (data not shown).