1 mM phenformin or 1 mM AICAr (AMPK stimulation), and
PEþphenformin or PEþAICAr (simultaneous hypertrophic
treatment and AMPK stimulation).- Incubate cells for 24 h in incubator at 37C, 5% CO 2.
Cell fixation
For this step, keep dishes on ice.- Rinse each well once with cold PBS.
- Add 2 ml of 4% PFAþ0.2% Triton X-100 for fixation during
30 min under the hood. - Remove dishes out from ice, remove PFA (seeNote 25), and
wash with PBS.
α-Actinin immunostaining - Wash coverslips 5 min with PBSþBSA 0.1%. Repeat this step
three times. - During these washing steps, prepare microscope slides. Draw
two circles per slide with dako pen. - Put 90μl of PBSþBSA 1% containing the anti-α-actinin
antibody in each drawn circle. - After removing the PBSþBSA 0.1% solution from each dish,
pick each coverslip and turn it over. Place each coverslip on its
corresponding droplet (seeNote 26). - Incubate for 1 h at room temperature.
- Put back each coverslip in the corresponding dish without
forgetting to turn it over (seeNote 27). - Wash coverslips 5 min with PBSþBSA 0.1%. Repeat this step
three times. - Remove excess of primary antibody solution. Wash the micro-
scope slides with PBSþBSA 0.1% and, then, with PBS. - Remove PBS and put 90μlofPBSþBSA 1% containing the
Alexa Fluor 594 donkey anti-mouse antibody in each drawn
circle. - After removing the PBSþBSA 0.1% solution from each dish,
pick each coverslip in the corresponding dish, and turn it over.
Place each coverslip on its corresponding droplet (seeNote 26). - Incubate for 1 h at room temperature and away from light.
- After 1 h, put back each coverslip in the corresponding dish
without forgetting to turn it over (seeNote 27). - Wash the coverslips three times with PBS and twice with dis-
tillated water. Keep away from light. - Wash the microscope slides once with PBS then once with
distillated water.
Study of Cardiac Hypertrophy and Protein Synthesis 335