In situ Hybridization of Embryos with Antisense RNA Probes - Caenorhabditis Elegans: Molecular Genetics and Development - page 253

T7 RNA Polymerase 1 m L

DEPC-ddH 2 O 3.5 m L (total volume: 10 m L)

Incubate in a thermocycler with a heated lid at 37 C, overnight. In the morning
add 30 m L DEPC-ddH 2 Oand0.5 m L RNase Inhibitor to the incubations and store
at 20 C.

IV. Animal Preparation and Fixation

A. Synchronization to Produce Gravid Hermaphrodites
Hermaphrodites must be synchronized for in situ experiments because the fixation
and freeze-crack techniques work best on animals that are similarly sized, rather than
a mixture of adults and larvae. In order to synchronize worms, two to three 10-cm
plates containing gravid hermaphrodites are bleached and the embryos are hatched
overnight in M9 + cholesterol [10 m g/mL] at 20 C. Synchronized L1 larvae are
collected by centrifugation and plated on 10-cm NGM plates for 3 days at 20 C.
After approximately 3 days the plates should contain gravid hermaphrodites with
early embryos. If later-staged embryos are required incubation may be extended a
further 5 h, but will depend on incubation temperature, abundance of food, general
health of the strain, and tendency of the worms to retain embryos.

B. Fixation of Gravid Hermaphrodites and Embryos
Harvest the worms by washing each 10-cm plate with 3 mL of M9 into several
Eppendorf tubes and centrifuging at 2000 rpm. This procedure often carries bacte-
rial contamination along with gravid hermaphrodites, depending on how much
bacteria remains on the plates. Excess bacteria will result in slides with high back-
ground after development. We routinely resuspend the worm pellet in 1 mL of
Hypaque meglumine (60%, available from http://www.nanric.com as Reno-60).
Invert the tube several times and centrifuge for 30 s at 2000 rpm. After centrifuga-
tion, the worms will float to the top of the Eppendorf tube ( Fig. 2A ). Remove the
worm layer and rinse 2-3 in M9. Examine an 80 m L droplet of worms on a
microscope slide under a dissecting microscope. If there are no bacterial clumps,
then the worms are clean and ready for freeze-cracking and fixation ( Fig. 2B ). If
many bacterial clumps are still present, repeat the meglumine flotation until worms
appear clean. Resuspend worms in 750 m LofM9.

C. Freeze-Cracking and Fixation
The following steps are carried out in RNase-free glassware, which should be
designated for RNA use and be rinsed with DEPC-ddH 2 O and baked for 4 h at
180 C. Alternatively, glassware may be cleaned thoroughly with RNase away (MBP,
#7003, 1L). Where indicated, disposable plastics may be used for convenience.