Carl confocal imaging system lsm 780

Cells were seeded on a special slide (Costar, Manassas, VA, USA). After 24 h, the cells on the slides were washed with PBS and 4% paraformaldehyde was added to fix the cells. After the cell membrane was permeabilized with 0.5% Triton X-100, the cells were blocked by adding 10% FBS for 30 min at room temperature. The primary antibody was added for incubation overnight at 4°C, and then the cells were washed three times with PBS, followed by the addition of a fluorescein isothiocyanate (FITC)-labeled secondary antibody (cat. no. A-21063; Invitrogen; Thermo Fisher Scientific, Inc.) and incubation for 1 h in a dark chamber. After washing three times with PBS, the cells were stained with DAPI for 5 min and analyzed with the Carl Zeiss Confocal Imaging System (LSM 780; Carl Zeiss, Jena, Germany).

To detect the expression and translocation of SMAD2/3, HCT116 cells which were prepared in cover glass were fixed for 10 min by 4% PFA. The cell membrane was permeabilized with 0.5% Triton X-100 for 10 min. After washing with 0.1% Tween20 in PBS [phosphate buffered saline (PBST)], cells were incubated with blocking buffer (5% bovine serum albumin, and 0.1% Triton-X 100 in PBS) for 1 h at room temperature. The primary antibodies against SMAD2/3 (1:200 dilution, Cell Signaling, Danvers, Massachusetts, USA), were incubated with fixed cells at 4°C overnight with gentle agitation. After washing completely with PBST, Alexa 594 conjugated goat antirabbit (1:5000 dilution, Life Technologies, Carlsbad, California) antibodies were added into a blocking buffer for 1 h at room temperature in a dark chamber. Cell nuclei were stained with DAPI (Vector Laboratories, Inc.; Burlingame, California, USA) for 2 min at room temperature. Fluorescence images were taken by the Carl Zeiss Confocal Imaging System (LSM 780; Carl Zeiss, Jena, Germany).