Immunohistochemistry
The C2C12 cells were reverse transfected with lipofectamine:DNA
complexes prepared according to the manufacturer’s protocol
(ThermoFisher, lipofectamine 3000). The C2C12 cells were trypsinized,
resuspended in media (DMEM+10%FBS, without antibiotics). The single
cell suspension was added to the lipofectamine mix at a concentration of
105 cells/well of 6-well dish. The cells were plated
on a 6-well plate and allowed to adhere overnight. Next day, the media
was changed to DMEM+10%FBS, with antibiotics. The cells were treated
with either DMSO or isoproterenol at the concentration of 1µM for 8
minutes. The cells were washed with 1XPBS, and fixed with 4%
formaldehyde overnight at 4°C. The cells were permeabilized with
1XPBS-0.1%Trition, 3X, 5’ each, followed by blocking in
1XPBS-0.1%Trition+10%FBS for 1 hour and overnight incubation in
primary antibody at 3 µgm/ml (Pde4D, catalog# PA521590 , Lot
#VH3049391A), followed by washes in 1XPBS-0.1%Trition, 3X, 5 minutes
each, and overnight incubation in secondary antibody with conjugated
Alexa-488. The cells were washed again in 1XPBS-0.1%Trition, 3X, 5 m
minute each and imaged with SP8 confocal microscope. The immunostaining
for p-Desmin for done as described above except for p-Desmin primary
antibody used at a concentration of 200 µgm /ml (p-Desmin Ser60,
ThermoFisher, Catalog# PA5-38837).