Biology Reference
In-Depth Information
Resuspend worms in the M9 by inversion. Pipette 80 m L of worm suspension onto
the center of a poly- L -lysine coated slide (Fisher, #12-550-19) and spread the droplet
with a clean razor blade so that the worms come in contact with the surface of the
slide. Begin cutting worms with the razor in an up-down motion while observing the
worms through the dissecting microscope. Stop when the majority of worms are cut
in half and the embryos are liberated. Starting at one side, cover the worms with a
22 40 mm cover glass (Gold Seal #3316), taking care not to introduce air bubbles.
While looking at the worms through the dissecting microscope, wick away excess
liquid by placing the edge of a Kimwipe or paper towel in contact with the edge of the
coverslip. This should be stopped just as adult worm carcasses cease to move but
before embryos burst open ( Fig. 4 ). As this step is critical for proper permeabiliza-
tion, it may need to be practiced until the researcher is confident that worms and
embryos have adhered properly to the slide. When a slide is ready, place it coverslip
side up on the aluminum disc and press down on the side that has no coverslip to
ensure complete contact. Incubate slides at least 5 min before proceeding to the
hydration series.
D. Hydration Series
Wedge a clean razor blade under one corner of the coverslip, and with a twisting
motion quickly pop off the cover glass in one motion. There should be an audible
''cracking'' sound as the coverslip is popped off. Incubate the slide as follows:
1) 5 min - 100% methanol (at 20 C)
2) 5 min - 90% methanol (room temperature)
3) 5 min - 70% methanol (room temperature)
4) 5 min - 50% methanol (room temperature)
5) 5 min - DEPC-ddH 2 O (room temperature)
6) 1 h - NTF (prewarmed to 37 C)
Fig. 4 Appearance of desired density of worms and embryos under coverslip, prior to freezing, as
viewed through a dissecting microscope.
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