Structure–Function Analysis of Yeast Tubulin - Methods in Cell Biology - page 358

created by this method. Alternately, a haploid strain containing GFP-TUB1 can be
crossed with haploid strains containing nonlethal tubulin mutations. The resultant
diploids are sporulated and dissected to generate control and experimental strains,
both containing an identical integration of GFP-TUB1 . If necessary, GFP-Tub1
levels among strains can be directly compared by western blot as described below.

22.5.3 Preparation of samples for microscopy
Prepare 5-ml overnight cultures at 30 C by inoculation from fresh plates into SC, or
appropriate SC-based selection media, with 0.2 mMadenine. The following day, dilute
cultures toOD600

0.02 in the samemedia and growwith shaking at room temperature
4-6 h to reach log phase. Pellet 1.5 ml for 1 min at 13,000 l

of fresh media. Sediment an additional 1.5 ml if a higher cell density is required for
imaging. Prepare a fresh sample from the log-phase culture after 20-30 min.
Note : Yeast strains auxotrophic for adenine ( ade ) require imaging media
supplemented with adenine to avoid high background fluorescence.

g and resuspend in30-50 m

Prepare microscope slides with agarose pads ( Fig. 22.4 ). Add 1.2% agarose in SC
media, boil briefly in a microwave, and pipet 40 l onto the center of a microscope

slide. Let stand for 2 s and place another slide on top of the agarose and let cool
2 min. Remove the top slide by gently rotating 60 and slowly sliding it away from
the bottom slide. Deposit 1.5

m

m l of resuspended yeast sample onto the pad and apply a

coverglass (22 1 mm). Do not apply strong pressure as this may damage the

cells. Seal the coverglass edges with melted valap to prevent drying during imaging.

22

Note I : To prepare valap, slowly melt ingredients (Vaseline, lanolin, and paraffin
1:1:1 w/w/w) using low setting on a hot plate and mix thoroughly using a
disposable heat-resistant container and stirring devise if possible. Pour into small
bottles, let cool, and cap for storage.
Note II : Valap is difficult to remove from microscope optics and will degrade
image quality. Do not allow valap to get onto microscope optics.
Note III : To apply, dip a wooden- or cotton-tipped applicator into melted valap
and spread quickly along the edges of the microscope coverglass.
Note IV : Use caution when heating valap for extended periods. The vapor will
resolidify on nearby surfaces.
Note V : Valap is only recommended for time-lapse imaging exceeding 2-3 min.

22.5.4 Microscopy and analysis of microtubule dynamics
Microtubules in live yeast are typically imaged with an automated microscope
equipped with a 63 Plan Fluor 1.4 N.A. objective, a filter set optimized for

GFP, and a cooled CCD camera. Typically, time-lapse series encompassing eight
z-series images with a step-size increment of 0.75 m m are captured at 8 s intervals.
To minimize photobleaching, the light source should be reduced using neutral den-
sity filters and the exposure time increased, yet still allow z-series to be acquired in