During late anaphase, Tem1 (green) localized to both spindle pole bodies (SPBs), but Bfa1 (red) was asymmetrically localized to the daughter SPB. DAPI (blue) and DIC (hidden) are also shown. Compare to metaphase image CIL# 13869. Image is Fig 6A, bottom panels, in J Cell Biol. (2011) 192: 599-614. Images in Fig 6 include CIL# 13869, 13868, 13871, 13870.
Cells (MATa tem1::TEM1-yEGFP::SpHIS5 bfa1::BFA1-mCherry-kanMX6) were fixed in 2.5% formaldehyde for 10 min, washed twice, and resuspended in 0.1 M potassium phosphate buffer, pH 6.4. Cells were then fixed for 10 min in 80% ethanol and resuspended in 1 mg/ml DAPI. Imaging was performed at 25C using a Leica DM6000 microscope equipped with a 100x/1.40 NA oil immersion objective lens, A4, L5, and TX2 filters, and a digital CCD camera (DFC350, Leica). Pictures were processed with LAS AF (Leica) and ImageJ software.
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