A 2-well Lab-Tek Chambered Borosilicate coverglass (Fisher Scientific) was coated with 20 mL of 0.1% (w/v) poly-L-Lysine solution (Sigma-Aldrich) for 30 min and dried. A yeast strain (Nup49p-GFP; Yeast GFP Clone Collection; Thermofisher) in which GFP was tagged to the nucleolar protein Nup49 was used. Stock solutions of Dil Stain (1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindocarbocyanine Perchlorate; Invitrogen), 250 μg/mL, and DAPI (200 μM) (Invitrogen) were prepared in dimethyl
sulfoxide and phosphate-buffered saline, respectively. Freshly isolated nuclei (20 μL) were mixed with 2 μL of Dil Stain and 2 μL of DAPI followed by incubation for 10 min at RT. Fluorescent stained nuclei were then applied to the coated cover glass and imaged with a Zeiss LSM 780 confocal microscope. Western blotting was performed using nuclei from W303 cells as described.12 (link) Anti-nop1p antibody (Santa Cruz Biotechnologies) was used as a nuclear marker at 1:500 dilution.
sulfoxide and phosphate-buffered saline, respectively. Freshly isolated nuclei (20 μL) were mixed with 2 μL of Dil Stain and 2 μL of DAPI followed by incubation for 10 min at RT. Fluorescent stained nuclei were then applied to the coated cover glass and imaged with a Zeiss LSM 780 confocal microscope. Western blotting was performed using nuclei from W303 cells as described.12 (link) Anti-nop1p antibody (Santa Cruz Biotechnologies) was used as a nuclear marker at 1:500 dilution.
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