3HA-Bfa1 (red) is generally localized to one spindle pole body (SPB) in wild-type cells in metaphase as determined by spindle morphology (tubulin, green) and nuclear morphology (DAPI, blue). Control image for CIL# 13891 in which constitutive targeting of Tem1 to both SPBs leads to symmetric SPB localization of Bfa1. Image is Fig 4C, top panels, in J Cell Biol. (2011) 192: 599-614. Images in Fig 4 include CIL# 13890, 13891, 13892, 13893.
Cells (MATa bfa1::3HA-BFA1) grown in rich media with glucose were fixed for 15 min in 3.7% formaldehyde and 0.1 M potassium phosphate buffer, pH 6.4. Cells were then washed twice with 0.1 M potassium phosphate buffer, pH 6.4, and resuspended in 1.2 M sorbitol in 0.12 M K2HPO4/0.033 M citric acid, pH 5.9. Fixed cells were digested with 0.1 mg/ml zymolyase-100T (US Biological) and 1/10 volume of glusulase (PerkinElmer) at 30C for 15 min, washed once, and resuspended in 1.2 M sorbitol in 0.12 M K2HPO4/0.033 M citric acid, pH 5.9. Primary antibodies were anti-HA monoclonal antibody (HA.11; 1:500) and anti-tubulin (Abcam; 1:500). Secondary antibodies were: anti-mouse Cy3 (for HA) and anti-rat FITC (for tubulin). Cells were resuspended in DAPI (1 mg/ml). 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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