The cells were washed twice with PBS,
fixed with 4% paraformaldehyde at 25 °C for 20 min, and then
washed in PBS containing 0.05% Tween 20. Nonspecific binding was blocked
by incubation of the cells with PBS containing 1% bovine serum albumin
(BSA). The cells were then incubated with primary antibodies to anti-α-amylase,
anti-Cytokeratin 7 and anti-ZO-1 (Santa Cruz Biotechnology), and anti-E-cadherin
(BD Bioscience) in a moist chamber overnight at 4 °C. After washing
in PBS, the cells were incubated with goat antimouse IgG-Alexa-488-conjugated
and goat antirabbit IgG-Alexa-555 secondary antibodies (Invitrogen)
for 2 h in the dark at 25 °C. Next, 4′,6-diamidino-2-phenylindole,
dihydrochloride (DAPI; Thermo Scientific) was added for 3–5
min to stain the cell nuclei. All experiments included a slide with
no primary antibody as a negative control. After mounting, cells were
viewed by using a confocal laser scanning microscope (Olympus FV1000,
Olympus, Tokyo, Japan).
fixed with 4% paraformaldehyde at 25 °C for 20 min, and then
washed in PBS containing 0.05% Tween 20. Nonspecific binding was blocked
by incubation of the cells with PBS containing 1% bovine serum albumin
(BSA). The cells were then incubated with primary antibodies to anti-α-amylase,
anti-Cytokeratin 7 and anti-ZO-1 (Santa Cruz Biotechnology), and anti-E-cadherin
(BD Bioscience) in a moist chamber overnight at 4 °C. After washing
in PBS, the cells were incubated with goat antimouse IgG-Alexa-488-conjugated
and goat antirabbit IgG-Alexa-555 secondary antibodies (Invitrogen)
for 2 h in the dark at 25 °C. Next, 4′,6-diamidino-2-phenylindole,
dihydrochloride (DAPI; Thermo Scientific) was added for 3–5
min to stain the cell nuclei. All experiments included a slide with
no primary antibody as a negative control. After mounting, cells were
viewed by using a confocal laser scanning microscope (Olympus FV1000,
Olympus, Tokyo, Japan).