Fm4 64 dye

Stage 12 flower bud was emasculated, and 18 h later the (stage 13–14E) pistil was cut transversally in the middle of the ovary and placed vertically in a half-cut, perforated PCR tube whose base was introduced into a solid agar medium. Pollen was deposited by gently touching the stigma with a mature anther. Incompatible pollen was deposited first, followed (within <1 min) by deposition of compatible pollen, which defined the timing starting point (T0) for monitoring pollen behavior. A cover slip was delicately applied on the surface of the pollinated stigma for confocal imaging. The system was maintained throughout the experiment at 21 °C and under 45% relative humidity. To increase the relative humidity in the vicinity of the pollinated stigma, pieces of solid agar medium were added around the mounted pistil. For cell surface labeling, after 35 min in high humidity conditions, pollinated stigma was incubated in FM4-64™ dye (N-3-triethylammoniumpropyl-4-6-4-diethylamino phenyl hexatrienyl pyridinium dibromide, Life Technologies T3166, 8.23 μM) for 5 min and subsequently washed in half-strentgth Murashige and Skoog basal medium containing 10% (w/v) sucrose, before mounting between a slide and cover slip in this medium.

To visualize vacuole morphology, the wild-type H99S strain and vps15Δ strains (YSB1500 and YSB1501) were cultured in liquid YPD medium at 30 °C for 16 h. FM4–64 dye (Life Technologies) was added to each culture at a final concentration of 10 μM and further incubated at 30 °C for 30 min. The cells were pelleted by centrifugation, resuspended with fresh liquid YPD medium, and further incubated at 30 °C for 30 min. The cells were pelleted again, washed three times with PBS, and resuspended in 1 ml of PBS. On the glass slide, 10 μl of the cells and 10 μl of mounting solution (Biomeda) were mixed and spotted. The glass slides were observed by confocal microscope (Olympus BX51 microscope).

HEK293A cells transfected with hHVRP1-EGFP or HVRP1*-EGFP in pNICE were grown on poly-D-lysine coated glass-bottom dishes (Mattek) for 1 to 2 days. Directly before imaging, live cells were washed in cold HBSS (Life Technologies) to remove residual FBS, and bathed in fresh HBSS. FM 4–64 dye (Life Technologies) was added to the solution (5 µM final concentration) to label the plasma membrane. Cells were imaged on a Zeiss LSM 780 confocal microscope with an LD C-Apochromat 63× immersion objective with 1.15 numeric aperture. For EGFP, excitation was at 488 nm, and emission band was 491–560 nm. For FM 4–64, excitation was at 561 nm, and emission band was 592–759. Primary cerebellar granule neurons growing on poly-D-lysine coated coverslips were fixated in 1∶1 methanol and acetone solution for ten minutes at −20°C. Immunocytochemistry was performed using either anti-HVRP1 antibody diluted 1∶1000, or anti-HVRP1 antibody diluted 1∶500 pre-incubated with peptide antigen (1∶500 dilution of a 1 µg/µl solution), and AlexaFluor-594-labeled goat anti-rabbit secondary antibody. Coverslips were then mounted onto Fisher SuperFrost Plus slides with ProLong Gold Antifade Reagent with DAPI (Life Technologies). For AlexaFluor 594, excitation was at 561 nm, and emission band was 592–759 nm. For DAPI, excitation was at 405 nm and emission band was 415–735 nm.