Cells were plated on a gelatin-coated chamber slide, transfected with
PQ-FRET sensors, fixed with 4% formaldehyde in CB (10mM MES pH 6.1,
138mM KCl, 3mM MgCl2, 2mM EGTA) containing 0.32M sucrose for 20 min,
rinse in TBS (20mM Tris-HCl pH7.4, 150mM NaCl), permeabilized with 0.5%
Triton X-100 in TBS for 10min, rinse in TBS-0.1%Tx (TBS containing
0.1% Triton X-100), blocked in 2% BSA in TBS-0.1%Tx, and
incubated with Alexa568-phalloidin (Invitrogen, 1:200 dilution) or primary
antibodies (mixture of mAb3–14 (5μg ml−1)
16 (link) and mAb1678
(5μg ml−1), Millipore) for 1 hr. After several washes
with TBS-0.1%Tx, the cells were incubated with secondary antibodies
(Invitrogen, 5μg ml−1), washed with
TBS-0.1%Tx, and mounted with mounting media (Spring Bioscience). Cells
were imaged using a spinning-disk confocal microscope as described above with a
60×/1.42 NA oil immersion objective (PLAPON 60XO, Olympus).
PQ-FRET sensors, fixed with 4% formaldehyde in CB (10mM MES pH 6.1,
138mM KCl, 3mM MgCl2, 2mM EGTA) containing 0.32M sucrose for 20 min,
rinse in TBS (20mM Tris-HCl pH7.4, 150mM NaCl), permeabilized with 0.5%
Triton X-100 in TBS for 10min, rinse in TBS-0.1%Tx (TBS containing
0.1% Triton X-100), blocked in 2% BSA in TBS-0.1%Tx, and
incubated with Alexa568-phalloidin (Invitrogen, 1:200 dilution) or primary
antibodies (mixture of mAb3–14 (5μg ml−1)
16 (link) and mAb1678
(5μg ml−1), Millipore) for 1 hr. After several washes
with TBS-0.1%Tx, the cells were incubated with secondary antibodies
(Invitrogen, 5μg ml−1), washed with
TBS-0.1%Tx, and mounted with mounting media (Spring Bioscience). Cells
were imaged using a spinning-disk confocal microscope as described above with a
60×/1.42 NA oil immersion objective (PLAPON 60XO, Olympus).
