At the end of the culture period, explants were fixed for 15 min in
4% paraformaldehyde and permeabilized with 5% Triton X-100 in
phosphate-buffered saline (PBS) after 3X PBS wash. Explants were then exposed to
a blocking solution containing 10% bovine serum albumin (BSA), and then
incubated with anti- myosin7A rabbit polyclonal antibodies (25–6790;
Proteus Bioscience Inc., Ramona, CA, USA; 1:500). Alexa-Fluor 568 goat
anti-rabbit IgG (A-11011, Invitrogen, CA, USA; 1:100) was used as the secondary
antibody. At the same time, specimens were incubated in FITC-conjugated or
Alexa-Fluor 633 phalloidin (1:100; Invitrogen) to label F-actin for 1 hr.
Finally, the explants were incubated with DAPI for 15 min. Specimens were
examined using a Leica TCS-SP2 laser-scanning confocal microscope (Leica
Microsystems Inc., Wetzlar, Germany). Cells with phalloidin-positive surface
structures were counted in randomly selected areas and averaged in each sample
for each condition, using an evaluation reticule 100 µm on each side.
Results from 6–7 samples were evaluated, based on initial observations
of variability.
4% paraformaldehyde and permeabilized with 5% Triton X-100 in
phosphate-buffered saline (PBS) after 3X PBS wash. Explants were then exposed to
a blocking solution containing 10% bovine serum albumin (BSA), and then
incubated with anti- myosin7A rabbit polyclonal antibodies (25–6790;
Proteus Bioscience Inc., Ramona, CA, USA; 1:500). Alexa-Fluor 568 goat
anti-rabbit IgG (A-11011, Invitrogen, CA, USA; 1:100) was used as the secondary
antibody. At the same time, specimens were incubated in FITC-conjugated or
Alexa-Fluor 633 phalloidin (1:100; Invitrogen) to label F-actin for 1 hr.
Finally, the explants were incubated with DAPI for 15 min. Specimens were
examined using a Leica TCS-SP2 laser-scanning confocal microscope (Leica
Microsystems Inc., Wetzlar, Germany). Cells with phalloidin-positive surface
structures were counted in randomly selected areas and averaged in each sample
for each condition, using an evaluation reticule 100 µm on each side.
Results from 6–7 samples were evaluated, based on initial observations
of variability.