Zymolyase

Cells were fixed in the growth medium with 3.7% formaldehyde at room temperature for 30 min and their cell walls digested with a solution containing 50 μg/ml zymolyase and 1:50 glusulase (Perkin Elmer). The digested cells were mounted on poly-L lysine coated slides to immobilize them for microscopy. Slide preparation for all samples was finished by adding mounting medium containing DAPI to stain the DNA.

Cells were fixed in growth medium with 3.7% formaldehyde at room temperature for 30 minutes. Mitotic cells were digested in SCE (1 M sorbitol/0.1 M sodium citrate/10 mM EDTA) with 20 μg/ml zymolyase (100T, ICN Biomedicals). Meiotic cells were digested with a solution containing 50 μg/ml zymolyase and 1:50 glusulase (Perkin Elmer). Cells were mounted on poly-L lysine coated slides and proteins detected by indirect immunofluorescence.
Immunostaining of whole cells was as follows: Cdc14-Myc7 fusion protein was detected with mouse monoclonal anti-Myc 9E10 antibody (Covance) at 1:600 in PBS/1% BSA for 3 hours at room temperature, followed by anti-mouse CY3 (Jackson ImmunoResearch) secondary antibody at 1:600 for 1 hour at room temperature. Slide preparation was finished by adding mounting medium containing DAPI. Detection of tubulin was done with the rat monoclonal antibody YOL 1/34, followed by anti-rat FITC (Jackson ImmunoResearch).