Imaging Embryonic Morphogenesis in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 400

18. Richardson ' s stain for thick sections. To prepare, make: A. 1% methylene blue
in 1% borax (w/v in dH20) and B. 1%Azure II in dH20. Mix equal volumes of A
and B and apply to sections with a syringe equipped with a syringe filter to
remove precipitates.
Media
1. Use 0.1 HEPES (N-Hydroxyethylpiperazine-N 0 -2-ethanesulfonate) buffer to pre-
pare agarose. A stock solution of 1MHEPES buffer is prepared by the addition of
a solution of HEPES acid to a solution of HEPES base (sodium salt): 13.01 g of
HEPES sodium salt is added to 50 mL of dH 2 O.
2. The freeze substitution medium is 1% osmium tetroxide with 0.1% uranyl acetate
in acetone ( McDonald, 1999 ). Briefly, to prepare 25 mL of 1% osmium with 0.1%
uranyl acetate, cool 24 mL of EM-grade acetone in a disposable 50 mL polypro-
pylene tube on crushed dry ice. If pure OsO 4 crystals are not consolidated in the
bottom of the vial, freeze the unopened vial of solid osmium in liquid nitrogen.
Osmium crystals will fall to the bottom of the ampule. Add 1 to 2 mL of the cold
acetone to the ampule, mix, and add back to the 50 mL tube of acetone on dry ice.
Repeat until all of the osmium is dissolved in 25 mL of acetone. Add the UA in
methanol (0.025 g UA in 1 mL of methanol) to the acetone, keep cold on dry ice.
Add 1 mL of freeze substitution mix (1% osmium tetroxide with 0.1% uranyl
acetate in acetone) to each 2 mL substitution vial and freeze in liquid nitrogen.
3. Methods
a. An Agarose Mount for Live Embryos
i. A thin (high-strength) agarose pad is formed over a standard glass micro-
scope slide. Agarose is dissolved in 0.1M HEPES (neutral pH) buffer to a
final concentration of 4% to 5%. The thickness of the pad can be controlled
by adding a single layer of cellulose tape over two slides on either side of the
slide to be coated. Add 100 m L of melted agarose to the top of the center slide
and compress the hot agarose to the thickness of a layer of Scotch tape
(approximately 60 m m) with a fourth slide resting on the two adjacent
tape-covered slides. Allow the agarose to solidify before sliding the top slide
off. Place the slide with the agarose pad in a humid chamber. A humid
chamber can be made by placing a 25-mm diameter Petri dish inside a 95-
mm Petri dish and adding water to cover the bottom of the large dish.
ii. C. elegans embryos are obtained by cutting open gravid hermaphrodites in a
watch glass filled with distilled water as described for standard agar mounts.
Only one group of approximately 10 embryos should be used. This grouping
typically fills the field of view when using a 60-63 objective. Larger
groups of embryos are impractical and difficult to navigate in the TEM.
iii. Place 70 m L of 5% low melting temperature agarose dissolved in 0.1M
HEPES along one edge of the agar pad. Quickly position a glass coverslip