- Incubate the membrane with primary antibody overnight at
4 C on a rotating device. - Then, wash the membrane with TBS-T twice for 5 min, once
for 10 min, and twice for 5 min on a stirring plate. - Incubate the membrane with the corresponding secondary
antibody diluted in TBS-T containing 5% (w/v) BSA for 1 h
at room temperature on a stirring plate. - After incubation, wash the membrane with TBS-T twice for
5 min, once for 10 min, and twice for 5 min on a stirring plate. - ECL solution mixture is prepared for the developing step, and
15 ml is placed on PVDF membrane. Shake membrane with
the ECL solution for 1 min. - Membrane is then placed between two plastic films and fixed in
an autoradiography cassette. - In the dark, a film is placed onto the membrane and the cassette
was closed. Open the cassette and remove the film after the
appropriate time of exposition. - Immerse the film in the developer buffer until the chemilumi-
nescence reaction occurs and you can detect a signal. Wash the
film with water and place it into the fixator buffer in order to fix
the chemiluminescence picture (Fig.2b, c , andf). - Film obtained can be quantified by software such as ImageJ
(Fig.2d)(seeNote 24).
3.4 Evaluation
of Cardiomyocyte
Hypertrophy by
α-Actinin
Immunostaining
Cell plating- During cardiomyocyte isolation (as presented before), wash
coverslips in ethanol and, then, in PBS, under the hood.
Place two coverslips per dish. - Add 2 ml of 0.2% gelatin solution in each dish for 15 min.
- Remove the excess and leave the dish open under the hood
until complete drying. - After cell counting using trypan blue, dilute cells in an adequate
volume of full IMDM knowing that 350,000 cells per dish are
needed for immunostaining analysis and wells can contain 2 ml
of medium. - Let cells to adhere overnight in incubator at 37C, 5% CO 2.
Cell treatments - The day after plating, medium is changed and replaced by 2 ml
of fresh full IMDM (pre-warmed at 37C). - Two days after plating, change and replace IMDM culture
medium with 2 ml of IMDM medium without serum
(pre-warmed at 37C) for 2 h before stimulation. - Cells are, then, treated according to the following conditions:
controls (no treatment), 20μM PE (hypertrophic treatment),
334 Florence Mailleux et al.