Te fm epi fluorescence system

GECS was plated onto coverslips coated with 0.1% (w/v) gelatin in a 24-well plate. The cells were fixed with 4% paraformaldehyde (PFA; Sigma-Aldrich, St. Louis, MO, USA) in phosphate-buffered saline (PBS) for 10 min and washed with PBS + 0.1% Tween-20 (PBST). Cells were blocked for nonspecific binding by incubation in 5% normal goat serum (NGS; Invitrogen, Waltham, MA, USA) in PBST for 30 min. Next, cells were stained for 30 min with the following primary antibodies: CD14, CD29, CD31, CD44, CD45, CD71, CD90, CD106, CD117, Sca-1 (1:200 dilution; all from BD Biosciences, San Jose, CA, USA), CD34, and CD133 (1:200 dilution; both from e-Bioscience, San Diego, CA, USA). Cells were stained with Alexa Fluor 594-conjugated secondary antibodies (1:1000 dilution; Molecular Probes, Eugene, OR, USA) for 30 min and washed three times in PBST. Nuclei were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma-Aldrich, St. Louis, MO, USA), and cells were mounted using fluorescent mounting medium (DAKO, Carpinteria, CA, USA). Fluorescence images were obtained using a TE-FM Epi-Fluorescence System attached to an Olympus BX61 inverted microscope (Olympus, Tokyo, Japan).

HUVECs were fixed with 2% PFA after treatment for 24 h with DMSO or KCH-1521, and blocked with 5% normal goat serum (NGS; #16210, Gibco) in 1× PBS + 0.1% Tween 20 (PBST) for 1 h. The cells were incubated for 1 h at room temperature (RT) with the following primary antibodies: talin (T3287), vinculin (V9131, both from Sigma-Aldrich), and paxillin (610051, BD Biosciences, San Jose, CA, USA). After washing, the cells were incubated for 1 h at RT with Alexa Fluor 594-conjugated anti-mouse IgG (A11005) and Alexa Fluor 488-conjugated Phalloidin (A12379, both from Molecular Probes, Eugene, OR, USA). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; D9542, Sigma-Aldrich). Negative controls for immunofluorescence staining were used to evaluate the specificity of primary antibodies and to exclude the possibility of non-specific staining of secondary antibodies by omitting the incubation of primary antibodies [37 (link)]. All immunofluorescent images were obtained using the TE-FM Epi-fluorescence system attached to an inverted microscope (BX61, Olympus, Tokyo, Japan).

Primary CSCs and hTERT-immortalized Sca-1+ CSC lines were plated onto coverslips coated with 0.1% (w/v) gelatin in 24-well plates. The cells were fixed with 4% paraformaldehyde in PBS for 10 min and washed with PBS + 0.1% Tween-20 (PBST). Cells were blocked for nonspecific binding by incubation in 5% normal goat serum (NGS; Invitrogen) in PBST for 30 min. Next, cells were stained for 30 min with the following primary antibodies: CD14, CD29, CD31, CD44, CD45, CD71, CD90, CD106, CD117, and Sca-1 (all from BD Biosciences), CD34 (e-Bioscience, San Diego, CA, USA), and CD133 (e-Bioscience). Cells were stained with Alexa Fluor 594-conjugated secondary antibodies (Molecular Probes, Eugene, OR, USA) for 30 min and washed three times in PBST. For control experiments, cells were stained with secondary antibodies only. Nuclei were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma-Aldrich, St. Louis, MO, USA), and cells were mounted using fluorescent mounting medium (DAKO, Glostrup, Denmark). Fluorescence images were obtained using a TEFM Epi-fluorescence system attached to an inverted microscope (Olympus, Tokyo, Japan) or were acquired with a confocal fluorescence microscope (LSM710, Carl Zeiss, Oberkochen, Germany).