Af6000 fluorescence microscope

Strains were grown in SD-Ura or SD-His medium overnight at 30°C. Cultures were diluted into 20 ml of SD medium (OD_{600 nm} 0.1) and grown to mid exponential phase (OD_{600 nm} 0.5–0.6). Cells were spun down and fixed immediately with 5 ml of 4% formaldehyde, 50 mM KPO₄ (pH 6.5) and 1 mM MgCI₂ for 2 h. After fixation, cells were washed in 5 ml of PM (0.1 M KPO₄ pH 7.5 and 1 mM MgCI₂) and resuspended in PMST (0.1 M KPO₄ pH 7.5, 1 mM MgCI₂, 1 M Sorbitol and 0.1% Triton X-100) to a final OD_{600 nm} of 10. 100 μl of cells were incubated with 0.6 μl of 2-mercaptoethanol and 1 mg ml⁻¹ zymolyase (Zymo Research, USA) for 40 min at 37°C. Spheroplast suspension was added to a polylysine-coated cover glass. The cells were blocked in 0.5% BSA/PMST for 30 min at RT, and incubated with primary antibody (6E10, Covance, USA) overnight at 4°C. After rinsing 3 times with PMST, cells were incubated with secondary antibody (anti-mouse Alexa 488, Dako, Denmark) for 2 h at RT in the dark. Then cells were stained with 0.4 mg ml⁻¹ DAPI (4′,6-diamidino-2-phenylindole) for 5 min in the dark. Images were acquired using Leica AF 6000 fluorescence microscope (Leica DMI4000B, Germany), and processed with LAS software.