- Allow these animals to reproduce, and evaluate their brood
size by counting the number of F 2 animals. - Once again, randomly select four unaffected plates, single
out 50 L4 F 2 larvae, let them reproduce, and evaluate their
brood size. - For all downstream analyses, we repeat these steps while
continuously selecting animals that are born from parents
that produce normal-sized broods.
3.3 Histone
Modifications
Identification by L1
Immunostaining Using
the Freeze/Crack
Method
Because of the epigenetic nature of the effect of acute starvation in
AMPK mutants, it may be of interest to assess the levels of various
histone marks at successive generations following the initial period
of starvation. If you choose to examine the levels of such modifica-
tions in the actual population that you have assessed for brood size
defects, it is important that your starting population is large enough
so that at each generation you obtain a minimum number of
C. eleganshermaphrodites to stain. The staining parameters are
well described in several standard immunostaining inC. elegans
procedures that make use of a typical freeze/crack step. For each
generation of interest, it will be essential to first collect gravid adult
hermaphrodites for treatment with alkaline hypochlorite (bleach-
ing) to obtain embryos and subsequently L1 larvae of generation of
interest.
To quantify the levels of various histone marks in the primordial
germ cells of L1 larvae or even in the germ cells of later stage larvae
at each generation, we use a standardC. elegansimmunostaining
procedure coupled with commercially available antibodies, or anti-
bodies that have been generously provided by ourC. eleganscol-
leagues or from the C. elegansGenetic Center (CGC) at the
University of Minnesota. The following steps will permit investiga-
tors to carry out the permeabilization and fixation procedures
followed by primary and secondary antibody binding steps.3.3.1 Preparing Slides
for Compound Microscopy
- Wipe pre-cleaned frosted edge slides with a lint-free tissue so
that they are very clean. - Spread a thin layer of the coating solution on the slides with a
cell rake, and let the slide dry for 10 min at 95C. Repeat the
operation twice.
3.3.2 Freezing Worms:
Preparing L1 Larvae
for Staining
- Prepare the L1 stock by performing alkaline hypochlorite treat-
ment (bleaching) as described above, and then let them
develop in M9 at 20C overnight. - The following day, wash the recovered L1 larvae two times with
water, and centrifuge the larvae at 400gfor 2–3 min (see
Note 15).
Quantifying Starvation-inducible Transgenerational Defects 575