Ab33533

CTCs were fixed with 4% paraformaldehyde (Santa Cruz Biotechnology) for 10 min and permeabilized with PBS containing 0.5% Triton X-100 for 5 min at RT. Nonspecific binding was blocked with 5% goat serum in PBS containing 0.1% Triton X-100 for 30 min at RT. Immunostaining was performed at 4 °C overnight with primary antibodies, followed by fluorescence-conjugated secondary antibodies at a concentration of 5 μg/mL (Thermo Fisher Scientific). Images were captured with the Zeiss LSM710 confocal microscope equipped with 64× NA 1.4 oil DIC (Carl Zeiss). Antibodies used for staining were as follows: rabbit anti-ALDH1 (bs-10162R, Bioss, 1:200), mouse anti-pan-CK (4545, Cell Signaling Technology, 1:3000), rabbit anti-EpCAM (ab71916, Abcam, 1:200), rabbit anti-NANOG (ab21624, Abcam, 1:800), mouse anti-OCT4 (ab184665, Abcam, 1:500), rat anti-CD44 (ab40983, Abcam, 1:400), mouse anti-CD45 (ab33533, Abcam, 1:200), goat anti-rabbit (A-11037), goat anti-rabbit (A-110340), and goat anti-mouse (A-11032).

Cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.5% Triton X-100 for 5 min at room temperature. Nonspecific binding was blocked with 5% goat serum in PBST (0.1% Triton X-100 in PBS) for 30 min at room temperature. Immunostaining was performed at 4 °C overnight with primary antibodies, followed by fluorescence-conjugated secondary antibodies (Invitrogen). Images were captured with Zeiss LSM710 confocal microscope at CSA Optical Imaging Facility. Primary antibodies used in immunofluorescence staining were as follows: mouse anti-γH2AX (1:400, 05–636, Millipore), mouse anti-pan-CK (1:3000, 4545, Cell Signaling Technology), rabbit anti-EpCAM (1:200, ab71916, Abcam), mouse anti-CD45 (1:200, ab33533, Abcam), rat anti-CD44 (1:400, ab40983, Abcam), rabbit anti-ALDH1 (1:200, bs-10162R, Bioss), rabbit anti-NANOG (1:800, ab21624, Abcam), mouse anti-OCT4 (1:500, ab184665, Abcam).

Cross-sections (7 μm cryosections from formalin-fixed samples) were washed in PBS (3 × 5 min), permeabilized in 0.5% Triton X-100 (30 min), and blocked for non-specific binding in 5% goat serum containing 1% BSA (30 min). The sections were then incubated with primary antihuman polyclonal antibodies against Ki67 (rabbit IgG, 1:200, Thermoscientific, RB-1510-P0), CD45 (mouse IgG1, 1:1000, Abcam, ab33533), vimentin (mouse IgM, 1:2000, Abcam, ab20346), αSMA (rabbit IgG, 1:600, Abcam, ab5694), or collagen I (mouse IgG1, 1:200, Sigma, c2456), in 10× diluted block solution (overnight at 4 °C). After washing in PBS (3 × 5 min), the sections were incubated with 1:500 secondary goat antibodies labeled with Alexa-488 conjugate (antimouse IgG1 (for CD45, Molecular Probes, A21121) or antirabbit IgG (for αSMA, Molecular Probes, A11008)) or Alexa-647 conjugate (antimouse IgM (for vimentin, Jackson Immunoresearch, 115-605-075) or antimouse IgG1 (for collagen I and Ki67, Molecular Probes, A21240)), in 10× diluted block solution (60 min). Nuclei were stained with 4′,6-diamidino-2-phenylindole (1:500, Sigma). The stained sections were mounted in mowiol (Sigma, 81381) and visualized with an inverted epifluorescent microscope (Zeiss Axiovert 200M, ×20/0.5 Plan-Neofluar lens, three sections/sample).