Dm2000 inverted microscope

Yeast cells were cultured to mid-log phase in the ironlimited medium. Harvested yeast cells were fixed in 5 ml of 50 mM KPO 4 (pH 6.5), 1 mM MgCl 2 and 4% formaldehyde for 2 h. After fixation, the cells were washed two times in 5 ml of PM buffer (100 mM KPO 4 pH 7.5, 1 mM MgCl 2 ) and followed by resuspension in PMST buffer (100 mM KPO 4 pH 7.5, 1 mM MgCl 2 , 1 M sorbitol, 0.1% Triton X-100) to a final OD600 of 10. 100 ll yeast cells were incubated for 20 min in 0.6 ll of b-mercaptoethanol and 1 mg/ml zymolyase (Zymo Research). The spheroplasted cells were washed with PMST buffer and attached to polylysine-coated coverslips. Adherent cells were blocked in PMST-BSA buffer (0.5% BSA in PMST buffer) for 30 min. Next, the adherent cells were incubated overnight at 4 °C with primary antibody (1:500 rabbit monoclonal ATP7B antibody, Abcam) diluted in PMST-BSA buffer. After incubation, the cells were washed three times with PMST-BSA buffer and incubated with secondary antibody (1:1000 anti-rabbit Alexa 488, Abcam) for 3 h at room temperature, and with 0.4 mg/ml DAPI (staining nuclei) for 5 min. Cells were mounted in Vectashield mounting medium (Vector Laboratories). Images were acquired using a Leica DM 2000 inverted microscope and processed with the Leica application suite (LAS-AF lite) software.