AMPK Methods and Protocols

3. Deposit 5–10μL of resuspended L1 larvae in water on to the
   coated slides (3–4 slides per condition, 2 spots containing L1
   larvae per slide).


4. Add 5μLof2fixation solution directly on the spot contain-
   ing the L1 larvae, and allow them to fix in a glass dish for 5 min
   at room temperature. Be sure the dish is humidified if longer
   incubations are used (alternatively this fixation step can be
   performed directly after the freeze/crack step with similar
   staining efficiencies). Place a metal block on dry ice during
   this period.


5. Gently place a coverslip over the spot of L1 larvae so that that
   the edge of the coverslip extends over the edge of the slide.
   Gently compress the two together, and remove any excess
   liquid with Whatman filter paper.


6. Without allowing the slide/coverslip to move or shift, carefully
   place the slides on the piece of chilled metal/metal block that is
   on dry ice. The slides should appear frozen within 1 min. Keep
   them on the metal block/dry ice for 5 min until they are
   thoroughly frozen (seeNote 16).

3.3.3 Freeze/Crack
and Fixation

1. For a better cracking result, remove the slides that were kept at
   
   80 C, allow to warm up on dry ice, and submerge them in a
   liquid nitrogen back just prior to the cracking step.


2. Crack the slide sandwich by swiftly separating the coverslip
   from the slide, and then immediately submerge the slide in a
   Coplin jar or a similar vessel filled with ice-cold methanol for
   1 min.


3. Air-dry the slides for 30 s to 1 min, and then submerge the
   slides in a Coplin jar filled with PBS for 5 min to rinse off the
   fixative. Wash two times with PBST, and proceed to the stain-
   ing procedure.

3.3.4 Immunostaining The antibody staining protocol is similar to standardC. elegans
protocols for any tissue [17].

1. Add 150μL of blocking solution per sample on the slides (two
   slots per slide), and allow 15 min–1 h in a humidified chamber.


2. Incubate each sample with 40μL of primary antibody (here
   rabbit anti-H3H4me3) solution diluted 1:1000 in blocking
   solution. Cover the sample in the depression in the slide with
   a piece of Parafilm, and leave it overnight in a moist chamber.


3. The following day, wash the slides three times in a jar contain-
   ing PBST, allowing at least 5 min between each wash.


4. Incubate the slide with secondary antibody diluted in blocking
   solution for 2–3 h. Add 40μL per sample, cover them with a

576 Emilie Demoinet and Richard Roy