Affinity Purification of Protein Complexes in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 300

Fig. 5 A) Early and late embryo preparation stained with Hoechst. B) Meiosis I arrested and endo-
mitotic chromosomes stained with Hoechst.
III.H. To determine the age of the embryos, about 5 m L of packed embryos are fixed
in 1 mL of cold methanol for at least 30 min, then incubated with 1 m g/mL Hoechst
stain in PBS+0.1% Triton X-100 for 10 min. After three washes with PBS+0.1%
Triton X-100, embryos are suspended in 30 m LPBS,and5 m Laremixedona
18 18 mm coverslip with 15 m L of mounting medium. The coverslip is carefully
placed on a slide, sealed with nail polish, and the number of nuclei per embryo is
determined using a fluorescence microscope ( Fig. 5A ).
J. Enriching for Meiosis I Arrested Embryos
Adult worms contain about 15 mitotically dividing embryos and at most two
meiotic embryos because meiosis is completed 30 min after fertilization. To enrich
for meiosis I metaphase, we used a mutation in a subunit of the anaphase-pro-
moting complex (APC). We chose a temperature-sensitive allele (g48)ofemb-27
( Cassada et al., 1981; Golden et al., 2000 ). At permissive temperature (16 C)
emb-27(g48) worms contain about 15 mitotically dividing embryos, similar to
wild type. At the restrictive temperature (24-25 C), emb-27(g48) embryos arrest
at metaphase of meiosis I and worms accumulate meiotically-arrested fertilized
embryos. Mutant worms are initially grown at 16 C as described in Section III.