Imaging Embryonic Morphogenesis in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 377

the mount. Embryos prepared with an agar mount are amenable to both light
microscopy (with differential interference contrast optics) or confocal microscopy.
Preparing C. elegans embryos on an agar mount is a simple technique that can easily
be mastered and is regularly done by undergraduates. It provides a consistent
embryonic orientation and environment that is suitable for long-term microscopy
of C. elegans embryos.

2. Other Mounting Methods
For many morphogenetic events, agar mounts are convenient because they pro-
duce uniform orientation of developing embryos. However, there may be times when
more randomized orientations are desired. Examples include imaging of the anterior
of the embryo during head enclosure, or events during gastrulation when an en face
view is desired. For these cases, other mounts are more useful. We discuss two types
here. Because protocols for producing these mounts are published elsewhere, we
only briefly mention them here.

a. Simple poly-L-lysine mount (see Mohler and Isaacson, 2010 for further details)

The simplest approach is to mouth pipette embryos in random orientations onto a
poly-L-lysine coated coverslip supported by grease feet above a microscope slide.

i. Spread a small volume of a 1 mg/mL stock of poly-L-lysine onto coverslips.
Allow the coverslips to air-dry for > 1 h. Poly-L-lysine is typically applied
from a premixed stock solution in distilled water (Sigma). Frozen stocks can
be aliquoted and stored at 20 C indefinitely. Avoid refreezing.
ii. Cut gravid hermaphrodites at the vulva in M9. Mouth pipette embryos in a
small volume (approx. 3 m L) of water onto precoated coverslips.
iii. Pipette a ring of silicon oil around the drop, and four dots of silicon vacuum
grease (Dow Corning) to the corners of the coverslip. It is typically conve-
nient to insert vacuum grease into a 10 mL plastic syringe without needle,
from which it can then be extruded. The grease ''feet'' provide a spacer that
allows a microscope slide to be affixed to the coverslip.
iv. Inverted a slide over the coverslip to form the mount. Press gently to allow
fluid to contact the slide.

b. Liquid mount with bead spacer (see Murray et al., 2006 for further details)
As an alternative to the simple poly-L-lysine mount method, polymer beads are
added to the medium to serve as spacers between the coverslip and the slide to
prevent the embryo from being excessively compressed. If the bead diameter is
< 25 m m (e.g., 20 m m), then this will result in slight compression of the embryos
and results similar to the standard Agar mount. If larger diameter beads are used
(e.g., 30 m m), then poly-L-lysine should be used as above.

i. Prepare a 1:30 dilution of 20 m m polymer beads (Polysciences, Inc.,
Warrington, PA; catalog #18329-5) mixed with M9 in a microfuge tube.