Biology Reference
In-Depth Information
due to their increasing radial spatial separation as they elongate, would appear as dis-
crete individual microtubules for imaging and analysis. Thus, the cut7.24
ts
monopolar
spindle would serve as the “control” background for analyzing individual spindle mi-
crotubule dynamics. A secondmutation can then be introduced into this background for
comparative studies of proteins which may regulate mitotic microtubules.
We detail here the step-by-step protocol for imaging individual spindle microtu-
bule dynamics in fission yeast.
24.1
METHODS
24.1.1
PDMS slide spacer
To prevent drying from long-term culturing and imaging at high temperature, we use
a relatively thick (
1 mm) pad of media infused agarose to position the fission yeast.
To have such a thick pad, a simple square well is created from the elastomer PDMS
(polydimethylsiloxane). To make the PDMS spacer:
1.
Pour liquid PDMS into a cup and then add curing agent for a final 9:1 ratio.
2.
Stir vigorously with a plastic fork for about 3 min to homogenize the mixture.
The stirring will generate small air bubbles, turning the clear mixture white.
3.
Put the cup in a vacuum desiccator for 30 min to remove all air bubbles. The
mixture should be again transparent at the end.
4.
Pour the mixture gently onto a large Petri dish to a height of
1 mm. Wait a
few minutes to allow any new air bubbles to reach the surface and gently blow
them away.
5.
Put the dish into an oven at 65
C for 2-4 h to cure and harden the mixture.
6.
After the PDMS mixture is cured or hardened, place a glass specimen slide on
top and using a surgical knife cut around the slide to get a slice of the same
exact size (25
75 mm). This piece should look like a slice of transparent and
flexible soft plastic.
7.
Cut out a square (
10
10 mm) at the center of the PDMS slice. This is now
ready for usage.
8.
The spacer is reusable after washing with warm soap and water.
Notes
On step 1, the ratio of PDMS to curing agent affects the stiffness of the
final PDMS product. More curing agent leads to stiffer PDMS products.
We find that 9:1 ratio is optimal for our work, but small changes (7:1-
12:1) will not be critical for this application.
On step 5, variation from the recommended 2-4 h curing time is not
critical for our application. However, too long a curing time could lead to
PDMS aging and brittleness. In this step is important to make sure that the
