Cd133

Tumor from all groups of mice were sectioned for histological studies. The tissue samples were fixed in 10% formalin and embedded in paraffin. The sections (5 μm) were cut using microtome, stained with hematoxylin and eosin, and slides were assessed using microscope (Leica microsystems, Germany) using at original magnification 10× and processed in Adobe Photoshop. For the immunofluorescence study, paraffin embedded sections were deparaffinized with xylene followed by rehydration with descending alcohol series. Slides were steamed with Reveal Decloaker (Biocare Medical), to minimize background staining, Sniper Universal Blocking Sera (Biocare Medical) was used throughout the protocol. Primary antibodies for Ki67 (Invitrogen), cd133 (Invitrogen) were diluted according to the manufacturer’s instruction and incubated overnight at 4 °C. Subsequently, Alexa Fluor-conjugated secondary antibody (Invitrogen) were used for visualizing. Slides were counterstained with DAPI and observed in a Leica fluorescent microscope. Negative control slides were used to discriminate nonspecific staining.

Canine peripheral blood samples (40 mL) were used to isolate mononuclear cells (MNCs) via canine peripheral blood mononuclear cell isolation kit (Solarbio, China) density gradient centrifugation, after which MNCs were cultured on fibronectin-coated T25 cell dishes containing EGM2 medium (EGM-2; Lonza, USA) at 37 °C in a 5%CO₂ humidified incubator. The phenotypes of ECFC were examined via flow cytometry using antibodies against mouse CD31, CD34, CD45 CD105, and CD133 (Invitrogen, USA). Dil-ac-LDL uptake and FITC-UEA-1 binding assays were conducted to further investigate the characteristics of ECFC.

A549/PTX protein samples were loaded onto a gel for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and run in 1 × Tris Glycine buffer (T&I, Korea). The proteins were transferred onto a nitrocellulose membrane (BIO-RAD, US). The membranes were incubated with primary antibodies against CD133 (Invitrogen), BMI-1 (Cell Signaling, USA), OCT3/4, SOX2, MUC1-C, β-catenin, PI3-K, p-AKT, and GAPDH (AbFrontier, Kroea) at 4°C overnight. Thereafter, the membranes were incubated with goat anti-rabbit IgG (Invitrogen, US) or mouse IgG (Santa Cruz Biotechnology, US) at 4°C overnight. Specific binding was detected using the SuperSignal chemiluminescent substrate (Thermo Fisher Scientific, US).

Tissues were deparaffinized by heating it at 56 °C overnight and then hydrated by dipping it in xylene (15 min, two times), 100% ethanol, 90% ethanol, 70% ethanol (two times), and PBS 5 min each. The slides were then steamed with a pH 9 reveal decloaker (BIOCARE Medical) for antigen retrieval, blocked in Dako serum-free blocker (Agilent technologies). Primary antibody against αSMA (Abcam), CD133 (Miltenyi), Ki67 (Invitrogen), CD206 (Abcam), CD8 (Abcam) was added overnight. Slides were washed 3× in PBS, secondary antibodies (Alexafluor-conjugated) were diluted in SNIPER (BIOCARE Medical), and slides were stained for 30 min at room temperature. Slides were washed again 3× in PBS and mounted using Prolong Gold anti-fade with DAPI (Thermo Fisher Scientific). Slides were dried overnight and imaged by fluorescent microscope. Images were acquired at ×20 and ×40.

Mouse intestinal tissue samples were collected and fixed in 10% formalin and were processed for paraffin embedding. Sectioned samples were immunostained according to standard protocols: samples were deparaffinized, blocked and incubated with primary, and fluorescence- or horseradish peroxidase (HRP)-conjugated secondary antibodies. For HRP-conjugated secondary antibody, 3,3'-Diaminobenzidine substrate was used, followed by haematoxylin nuclear counterstaining. Antibodies against the following were used for immunohistochemistry: LIG4 (Sigma (HPA001334); 1:250 dilution); phospho-γH2AX (Cell Signaling (20E3); 1:250 dilution); RFP (Thermo (RF5R); 1:250 dilution); cleaved caspase 3 (Cell Signaling (5A1E); 1:500 dilution); β-catenin (Cell Signaling (D10A8); 1:500 dilution); CD44 (BD Pharmingen (G44-26); 1:250 dilution); and CD133 (eBioscience (13A4); 1:200 dilution). Immunostained tissues were photographed using a dissection microscope (Zeiss; AxioVision). For comparison, images were captured with the same exposure time. CRC tissue microarray slides were purchased from Biomax (Co2086).

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).

The CD mouse antibodies used in the antibody screen were the following CD antibodies conjugated with PE from BD Biosciences: CD1d (553846), CD2 (553112), CD3 (555275), CD5 (553022), CD8a (553032), CD11b (553311), CD11c (553802), CD13 (558745), CD14 (553740), CD15 purified (559045), CD15-APC conjugated (551376), CD18 (553293), CD19 (553786), CD23 (553139), CD24 (553262), CD25 (553866), CD27 (558754), CD28 (553297), CD30 (553825), CD31 (553373), CD38 (553764), CD40 (553791), CD43 (553271), CD44 (553134), CD49b (558759), CD49d (557420), CD49e (557447), CD51 (551187), CD54 (553253), CD55 (558037), CD61 (553347), CD62e (553751), CD69 (553237), CD80 (553769), CD81 (559519), CD86 (553692), CD90 (554898), CD90.2 (553005), CD95 (554258), CD103 (557495), CD117 (553355), CD121a (557489), CD121b (554450), CD122 (553362), CD124 (552509), CD126 (554462), CD127 (552543), CD131 (559920), CD133 (eBioscience/Thermo-Fisher; 12–1331), CD24 unconjugated (BD 557436), CD133 (eBioscience 14-1331-80).