Plan apochromat 100x 1.4 oil dic m27 objective

Yeast cells were grown as above for immunoblotting. Upon harvesting, cells were fixed in 4% paraformaldehyde by incubating at room temperature for 15 min, and washed again in 1X PBS. Cells were then immobilized onto polylysine coated coverslips (Neuvrito) and mounted with ProLong™ Diamond antifade mountant with DAPI (Thermo Fisher). Fluorescence microscopy for quantification was acquired using a Zeiss Axio observer.Z1 inverted microscope equipped with a Plan-Apochromat 100x/1.4 oil DIC M27 objective (Zeiss), X-cite 120 LED system (Lumen Dynamics), filter set (HE) RFP/GFP/DAPI (Zeiss) and a digital Axiocam MRm camera (Zeiss) controlled with the Zen blue software. Raw data was collected as z-stacks and projected using ImageJ (NIH), and quantification was performed manually. Confocal microscopy of representative cells was collected on a Leica TCS SP8 inverted sSTED microscope equipped with a 100x/1.40 APO objective and using the following detection mirror settings: RFP 560–630nm, GFP 470–536. DAPI staining was detected using a blue diode 405nm laser (10%) and a photon multiplying tube. One representative middle slide was collected, and images were subsequently deconvolved and background subtracted using Huygens Professional (Scientific Volume Imaging).