piece of Parafilm as above, and leave them in a dark humid
chamber for 2–3 h at room temperature.- Wash the slides three times in a jar containing PBST allowing at
least 5 min between each wash. - Add DAPI (1μg/mL in PBST) to the slide to counterstain the
nuclei, incubate 2 min, and rinse quickly. Alternatively, DAPI
can be added to the mounting solution. - Remove most of the liquid, and add the mounting solution
(i.e., Vectashield or ProLong antifade). Seal the edges with nail
polish if you need to maintain the slide for future analysis. - The samples can then be imaged using a compound or a
confocal microscope equipped with the appropriate filters and
wavelengths. Following the imaging the slides can be main-
tained at 80 C for an extended period of time.
Although at present we can conclude that functional AMPK is
required to ensure that such epigenetic modifications that are
typical of activating gene expression in the germ line do not occur
during periods of acute starvation, i.e., during the L1 diapause, we
believe that AMPK may also have other critical roles in adjustment
that contribute to these transgenerational defects in brood size. By
performing simple, well-organized multigenerational lineages,
investigators that are interested in identifying novel AMPK targets
can pinpoint the key processes that may be affected in the progres-
sive deterioration in reproductive fitness that we observe in these
mutant animals following a brief period of starvation. We envisage
that these may include effectors of small RNA biosynthesis, other
histone modifying enzymes, and/or metabolic enzymes or precur-
sors required to generate critical substrates for all these AMPK-
regulated processes.4 Notes
- Prepare the 2fixative solution, and use as soon as chilled or
later the same day. Discard any unused fixative in the appropri-
ate disposal. - Be careful when you start your experiment: animals need to be
freshly thawed and grown at least three generations at 20C
without being starved at any time. - This typically takes between 7 and 9 min, but the duration will
depend on the starting volume of pellet, temperature, and the
efficiency of the hypochlorite solution. - The embryos are more resistant to this treatment, but don’t let
the reaction go too long, or all the embryos will die as well.
Quantifying Starvation-inducible Transgenerational Defects 577