Triton X-100 (0.1% in 1× PBS)
(Sigma) was used to permeabilize cells, then blocked for 30 min with
1% BSA (Roche, Switzerland), 22.5 mg/mL glycine, and 0.1% Tween 20
(Sigma) in PBS. The following primary antibodies were used:
To visualize sarcomeric myosin expression, anti-MF20 (contributed
to Developmental Studies Hybridoma Bank by Fischman, D.A.70 (link)) was diluted 1:2 in 1% BSA in PBS with 0.1%
Tween 20 (dilution buffer) and incubated overnight at 4 °C. For
1-day experiments, phosphor-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) primary
antibody (Cell Signaling Technology) or YAP (Santa Cruz Biotechnology)
were diluted 1:200 in dilution buffer and incubated at 4 °C overnight.
Bound antibodies were then fluorescently tagged for 1 h at room
temperature with species-appropriate secondary antibodies (for myosin,
goat anti-mouse Alexa555 secondary antibody (Molecular Probes); for
phosphor-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), goat anti-rabbit Alexa555
(Molecular Probes); for YAP, goat anti-mouse Alexa647 (Abcam)) diluted
1:1000 dilution buffer. Cells were washed three times in 1× PBS
after each step. All surfaces were counterstained with NucBlue (Molecular
Probes) for 20 min at room temperature to visualize nuclei, and MF20-stained
cells were also counterstained with Phalloidin-iFluor 647 (Abcam)
to visualize actin. The cells were then imaged at 20× magnification.
(Sigma) was used to permeabilize cells, then blocked for 30 min with
1% BSA (Roche, Switzerland), 22.5 mg/mL glycine, and 0.1% Tween 20
(Sigma) in PBS. The following primary antibodies were used:
To visualize sarcomeric myosin expression, anti-MF20 (contributed
to Developmental Studies Hybridoma Bank by Fischman, D.A.70 (link)) was diluted 1:2 in 1% BSA in PBS with 0.1%
Tween 20 (dilution buffer) and incubated overnight at 4 °C. For
1-day experiments, phosphor-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) primary
antibody (Cell Signaling Technology) or YAP (Santa Cruz Biotechnology)
were diluted 1:200 in dilution buffer and incubated at 4 °C overnight.
Bound antibodies were then fluorescently tagged for 1 h at room
temperature with species-appropriate secondary antibodies (for myosin,
goat anti-mouse Alexa555 secondary antibody (Molecular Probes); for
phosphor-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), goat anti-rabbit Alexa555
(Molecular Probes); for YAP, goat anti-mouse Alexa647 (Abcam)) diluted
1:1000 dilution buffer. Cells were washed three times in 1× PBS
after each step. All surfaces were counterstained with NucBlue (Molecular
Probes) for 20 min at room temperature to visualize nuclei, and MF20-stained
cells were also counterstained with Phalloidin-iFluor 647 (Abcam)
to visualize actin. The cells were then imaged at 20× magnification.