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Yeasts were incubated protected from light with 5 M TopFluor Cholesterol (810255, Avanti Polar Lipids) for 2 h at 20 C

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Volume electron microscopy Cells grown on glass-bottom dishes with etched grid markings (MatTek, Ashland, MA) were fixed with 2% glutaraldehyde (Sigma-Aldrich) in 0.1 M sodium cacodylate, 2 mM CaCl 2 buffer (pH 7.4), for 30 min at room temperature and stained with LD540 (0.1 g/mL) in 0.1 M sodium cacodylate buffer for 20 min

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