For microscopy, cells were cultured in 35 mm glass-bottom Petri dishes (Greiner Bio-One International).
For live cell confocal microscopy, cells were seeded onto 50 mm PDL-coated glass bottom plates (Warners Instruments).
For electron microscopy cells were fixed in 1% OsO4 and 5% glutaraldehyde in filtered (0.2µm) seawater in centrifuge glasses for about 15 min". "The cells measured 4 5µm.
For immunofluorescence microscopy, cells were grown in CY medium until OD600 = 0.3 and fixed for 15 min at room temperature and 45 min on ice in 4% (wt/vol) paraformaldehyde.
For analysis of expression levels by microscopy, cells were fixed in 4% PFA, probed with anti-CXCR4 antibody (1 100) (Sigma C8352 or ThermoFisher Scientific, Cat No. MHCXCR404) for 1 h, followed by TRITC conjugated IgG antibody (anti-rabbit, 1 400).
For live-cell confocal microscopy, cells were visualized with a 100×/1.46 oil immersion objective using the Zeiss LSM 510 confocal microscope equipped with a TempModuleS and CO2 Module (Zeiss) to maintain the temperature and CO2 levels at 37 °C and 5.0%, respectively.
For thin-section electron microscopy, cells were fixed in 2.5% glutaraldehyde, 2% paraformaldehyde and 0.1% picric acid in 100 mM phosphate (pH 6.5) for 2 h at 4 °C followed by post-fixation in 1% osmium tetraoxide in 100 mM phosphate buffer (pH 6.5) with 50 mM sucrose for 1 h at 4 °C.
To visualize RFP expression using fluorescent microscopy, cells were rinsed twice with PBS and fixed using ProLong Gold Antifade Reagent with 4′,6-diamidino-2-phenylindole (DAPI Life Technologieses).
For fluorescence microscopy, cells were grown to OD600 = 0.8 − 1.0 in appropriate selective medium and shifted to SD-N medium for various lengths of time as described (Cheong et al., 2005).
Before microscopy cells were washed several times with YPD.
For immunoelectron microscopy, cells were processed by freeze substitution.
