Biology Reference
In-Depth Information
to GFP. Since phalloidin staining of F-actin containing structures is such an
important technique for studying epidermal morphogenesis, we provide this alter-
native procedure to that described by Maddox and Maddox in Chapter XX. An
example of an embryo stained with phalloidin to reveal actin-rich structures is
shown in
Fig. 4C
.
(a) Phalloidin staining for analyzing morphogenesis
Method
1. Make solutions and coat ring slides with 25
m
L poly-L-lysine.
2. Obtain eggs from adults by bleaching. Make sure to sweep embryos off the
plate using a glass pipette.
a. Hint: It is sometimes advantageous to bleach embryos for a slightly
longer period of time to further weaken the eggshell. This can be done
by allowing the embryos to remain in the bleach solution for another
minute after the worm carcasses have all been dissolved.
3. Wash embryos at least 2
with ddH
2
O. We find they stick to the slide better
when using water, not M9.
4. Add embryos to ring slides that have been precoated with poly-L-lysine. Let
sit 5-10 min to give embryos time to adhere.
a. Hint: It helps the embryos to adhere when the slide is rinsed with ddH
2
O
before adding the embryos.
5. Remove the water from the ring slide. The bulk of the liquid can be poured
off into a liquid waste container, if care is taken to avoid mixing embryos
from one ring to another. Use a Kimwipe to gently remove the remaining
liquid.
6. Add 45-60
m
L of the fix/permeabilization solution to each ring.
a. Incubate the slides for 20 min in a humid chamber at room temperature.
If there are many embryos, it is advisable to incubate 2-5 min longer.
b. A simple humid chamber can be made by placing wet paper towels in the
bottom of a Tupperware container.
7. Wash slides 2
5 min with 1
PBS by adding 70
m
L of PBS to each ring
per wash. Remove liquid after each wash as above.
8. Add 60
m
L phalloidin solution to each ring.
a. Incubate 1-2 h in a humid chamber at room temperature in the dark (we
usually incubate for 90 min). Alternatively, incubations can be carried
out at 4
C overnight in the dark, which can reduce background.
9. Wash slides 2
5 min with 1
PBS while rotating.
10. Dry off slide using a Kimwipe. Mount by inverting an 18 mm square
coverslip with 8
m
L SlowFade over each sample. Seal edges with nail
polish.
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