Journal: PeerJ

Article Title: DNA mismatch repair and CD133-marked cancer stem cells in colorectal carcinoma

doi: 10.7717/peerj.5530

Figure Lengend Snippet: (A) The weak CD133 staining seen at the luminal border of some normal colonic crypts is considered equivocal and interpreted as negative (x200). In contrast, (B) is a case of colorectal carcinoma with strong CD133 staining of the glandular luminal surface (x200) and (C) a case of poorly differentiated colorectal carcinoma demonstrating CD133 staining as tiny cytoplasmic blobs which represent abortive glands (x200). (D) Colorectal carcinoma with deficient DNA mismatch repair (represented by loss of MLH1). Note the lack of immunoexpression of MLH1 in the colorectal carcinoma nuclei in the presence of immunopositivity in the internal positive controls (stromal cells, lymphocytes and nearby normal enterocytes) (x200)

Article Snippet: Immunohistochemical staining with a rabbit polyclonal antibody to CD133 (1:200; Abcam ab19898, Cambridge, United Kingdom) and detection by the ultraView universal DAB detection kit (Ventana Medical Systems Inc., Tucson, AZ, USA) was carried out on the Ventana Benchmark XT autostainer (Ventana Medical Systems Inc., Tucson, AZ, USA).

Techniques: Staining

Journal: PeerJ

Article Title: DNA mismatch repair and CD133-marked cancer stem cells in colorectal carcinoma

doi: 10.7717/peerj.5530

Figure Lengend Snippet: CD133 expression and DNA mismatch repair (MMR) status. The CD133 expression and MMR status as per size, TNM stage and differentiation of the colorectal carcinoma ( n = 80).

Article Snippet: Immunohistochemical staining with a rabbit polyclonal antibody to CD133 (1:200; Abcam ab19898, Cambridge, United Kingdom) and detection by the ultraView universal DAB detection kit (Ventana Medical Systems Inc., Tucson, AZ, USA) was carried out on the Ventana Benchmark XT autostainer (Ventana Medical Systems Inc., Tucson, AZ, USA).

Techniques: Expressing

Journal: PeerJ

Article Title: DNA mismatch repair and CD133-marked cancer stem cells in colorectal carcinoma

doi: 10.7717/peerj.5530

Figure Lengend Snippet: DNA mismatch repair protein (MMR) status and CD133 expression. CD133 immunoexpression and score being stratified by MMR status in right-sided and left-sided colorectal carcinomas.

Article Snippet: Immunohistochemical staining with a rabbit polyclonal antibody to CD133 (1:200; Abcam ab19898, Cambridge, United Kingdom) and detection by the ultraView universal DAB detection kit (Ventana Medical Systems Inc., Tucson, AZ, USA) was carried out on the Ventana Benchmark XT autostainer (Ventana Medical Systems Inc., Tucson, AZ, USA).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Sensitivity of Human Intrahepatic Cholangiocarcinoma Subtypes to Chemotherapeutics and Molecular Targeted Agents: A Study on Primary Cell Cultures

doi: 10.1371/journal.pone.0142124

Figure Lengend Snippet: Sequences of primer pairs (sense and antisense, respectively) used for amplifying the genes of interest (GOI) and the internal reference gene (GAPDH) used for their nor Primer designed by the PROBEFINDER software ( https://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp ).

Article Snippet: Cells were rinsed twice with PBS buffer for 2 min, blocked and incubated 1 hour with the following antibodies: Vimentin mouse monoclonal IgG1 (sc-32322, Santa Cruz Biotechnology), α-SMA mouse monoclonal IgG1(M0851, Dako), E-Cadherin mouse monoclonal IgG1 (sc-21791, Santa Cruz Biotechnology), CD326/EpCAM mouse monoclonal IgG1 (sc-59782, Santa Cruz Biotechnology), CD133/PROM1 mouse monoclonal IgG1 (TA309943, Origene), LGR5 rabbit polyclonal IgG (TA301323,Origene), cytokeratin-19 (CK-19) sc-6278 CK-19 mouse monoclonal IgG2a (Santa Cruz Biotechnology), Interleukin 6 (IL6) mouse monoclonal IgG2a (ab9324, Abcam) at room temperature.

Techniques: Software, Amplification

Journal: PLoS ONE

Article Title: Sensitivity of Human Intrahepatic Cholangiocarcinoma Subtypes to Chemotherapeutics and Molecular Targeted Agents: A Study on Primary Cell Cultures

doi: 10.1371/journal.pone.0142124

Figure Lengend Snippet: (A) Immunohistochemical and immunofluorescence analyses of mixed- and mucin-IHCCA primary cultures for Vimentin, α-SMA, E-Cadherin and the “epithelial” cancer stem cell markers CD133+, EpCAM+, LGR5+ and for IL6. A diffuse positivity for mesenchymal markers (Vimentin, α-SMA) and IL6 was observed while E-Cadherin was virtually negative and less than 5% cells where positive for the “epithelial” cancer stem cell markers. Representative experiment of N = 12 independent staining performed in separate primary cultures. (B) Flow cytometry analyses of primary cultures of mixed- and mucin-IHCCA (20–30 passages), labeled with anti-CD13, anti-CD90, anti-EpCAM, anti-CD133, and anti-LGR5 antibodies. Bar graphs and representative plots. Cells positive for CD13 and CD90 largely predominated with respect to CD133, EpCAM and LGR5. CD13+ cells predominated in mixed-IHCCA with respect to mucin-IHCCA, while the opposite was found for CD90+ cells. Mean ± SD of N = 18 independent experiments. * = p< 0.05 vs mucin-IHCCA; & = p< 0.01 vs mixed-IHCCA.

Article Snippet: Cells were rinsed twice with PBS buffer for 2 min, blocked and incubated 1 hour with the following antibodies: Vimentin mouse monoclonal IgG1 (sc-32322, Santa Cruz Biotechnology), α-SMA mouse monoclonal IgG1(M0851, Dako), E-Cadherin mouse monoclonal IgG1 (sc-21791, Santa Cruz Biotechnology), CD326/EpCAM mouse monoclonal IgG1 (sc-59782, Santa Cruz Biotechnology), CD133/PROM1 mouse monoclonal IgG1 (TA309943, Origene), LGR5 rabbit polyclonal IgG (TA301323,Origene), cytokeratin-19 (CK-19) sc-6278 CK-19 mouse monoclonal IgG2a (Santa Cruz Biotechnology), Interleukin 6 (IL6) mouse monoclonal IgG2a (ab9324, Abcam) at room temperature.

Techniques: Immunohistochemical staining, Immunofluorescence, Staining, Flow Cytometry, Labeling

Journal: PLoS ONE

Article Title: Sensitivity of Human Intrahepatic Cholangiocarcinoma Subtypes to Chemotherapeutics and Molecular Targeted Agents: A Study on Primary Cell Cultures

doi: 10.1371/journal.pone.0142124

Figure Lengend Snippet: RT-PCR analysis of Vimentin and cancer stem cell surface markers.

Article Snippet: Cells were rinsed twice with PBS buffer for 2 min, blocked and incubated 1 hour with the following antibodies: Vimentin mouse monoclonal IgG1 (sc-32322, Santa Cruz Biotechnology), α-SMA mouse monoclonal IgG1(M0851, Dako), E-Cadherin mouse monoclonal IgG1 (sc-21791, Santa Cruz Biotechnology), CD326/EpCAM mouse monoclonal IgG1 (sc-59782, Santa Cruz Biotechnology), CD133/PROM1 mouse monoclonal IgG1 (TA309943, Origene), LGR5 rabbit polyclonal IgG (TA301323,Origene), cytokeratin-19 (CK-19) sc-6278 CK-19 mouse monoclonal IgG2a (Santa Cruz Biotechnology), Interleukin 6 (IL6) mouse monoclonal IgG2a (ab9324, Abcam) at room temperature.

Techniques:

Journal: Scientific Reports

Article Title: Screening of aptamers specific to colorectal cancer cells and stem cells by utilizing On-chip Cell-SELEX

doi: 10.1038/srep10326

Figure Lengend Snippet: ( a ) CR-CSCs form suspension cultured tumorspheres from culture day one to day five; ( b ) anti-CD44 fluorescence immunostaining analysis with CR-CSCs after enrichment suspension culture and colorectal cancer cells (HCT-8); ( c ) anti-CD133 fluorescence immunostaining analysis with CR-CSCs after enrichment suspension culture and HCT-8.

Article Snippet: USA.) was applied for CD44 staining and a CD133 antibody (C24B9, Rabbit, Cell Signaling Tech.

Techniques: Suspension, Cell Culture, Fluorescence, Immunostaining

Journal: The American Journal of Pathology

Article Title: Profiles of Cancer Stem Cell Subpopulations in Cholangiocarcinomas

doi: 10.1016/j.ajpath.2015.02.010

Figure Lengend Snippet: List of Antibodies Used for IHC and IF

Article Snippet: For all immunoreactions, negative controls (the primary antibody was replaced with preimmune serum) were also included. shows the details of antibodies used in the study. table ft1 table-wrap mode="anchored" t5 caption a7 Name Host/isotype Source Catalog# Dilution CD326/EpCAM Mouse IgG1 Santa Cruz Biotechnology (Dallas, TX) sc-59782 1:50 K7 (cytokeratin 7) Mouse IgG1 Dako M7018 1:100 K19 (cytokeratin 19) Mouse IgG1 Abcam (Cambridge, UK) ab87014 1:50 K19 (cytokeratin 19) Mouse IgG1 Dako M0888 1:100 HepPar-1 Mouse IgG1 Dako M7158 1:50 CD133/Prominin 1 Rabbit IgG Abnova (Taipei, Taiwan) {"type":"entrez-protein","attrs":{"text":"PAB12663","term_id":"1236625334","term_text":"PAB12663"}} PAB12663 1:100 CD133/PROM1 Mouse IgG1 OriGene (Unimed Scientifica, Rome, Italy) TA309943 1:50 CD90/Thy1 Rabbit IgG Abcam ab92574 1:100 CD13 Mouse IgG1 Novacastra Reagents (Leica Biosystems, Buffalo Grove, IL) NCL-CD13-304 1:100 LGR5 Goat IgG Santa Cruz Biotechnology SC-68580 1:50 Desmin Mouse IgG1 Dako M0760 1:100 Vimentin Mouse IgG1 Santa Cruz Biotechnology sc-32322 1:100 Nestin Mouse IgG1 Santa Cruz Biotechnology sc-23927 1:100 α-SMA Mouse IgG1 Dako M0851 1:50 S100A4 Rabbit IgG Dako A5114 1:100 SNAIL Rabbit IgG Santa Cruz Biotechnology sc-28199 1:50 TWIST Rabbit IgG Santa Cruz Biotechnology sc-15393 1:50 LGR5 Rabbit IgG OriGene TA301323 1:50 NCAM-PE Mouse IgG1 BD Pharmingen (Milan, Italy) 555.516 1:50 E-cadherin Mouse IgG1 Santa Cruz Biotechnology sc-21791 1:50 P-cadherin Rabbit IgG Santa Cruz Biotechnology sc-7893 1:50 GFAP Mouse IgG1 Dako M0761 1:50 CD163 Mouse IgG1 OriGene TA506382 1:50 CD31 Mouse IgG1 Dako M0823 1:50 CD13 Mouse IgG1 Abcam Ab7417 1:100 SDF1 Rabbit IgG Santa Cruz Biotechnology sc-28876 1:50 FAP Mouse IgG1 Santa Cruz Biotechnology sc-65398 1:50 Periostin Goat IgG Santa Cruz Biotechnology sc-49480 1:50 Periostin Rabbit IgG Santa Cruz Biotechnology sc-67233 1:50 CD90-FITC Human Miltenyi Biotec (Cologne, Germany) 130-095-403 1:10 CD326 (EPCAM)-FITC Human Miltenyi Biotec 130-080-301 1:10 Goat anti-rabbit FITC IgG Abcam ab-6717 1:400 Goat anti-rabbit TRITC IgG Abcam ab-6718 1:400 Goat anti-mouse FITC IgG Abcam ab-6785 1:400 Goat anti-mouse TRITC IgG Abcam ab-6786 1:400 Open in a separate window List of Antibodies Used for IHC and IF Sections were examined in a coded fashion by Leica Microsystems DM 4500 B Light and Fluorescence Microscopy (Leica Microsystems, Weltzlar, Germany) equipped with a Jenoptik ProgRes C10 Plus Videocam (Jenoptik, Jena, Germany).

Techniques:

Journal: The American Journal of Pathology

Article Title: Profiles of Cancer Stem Cell Subpopulations in Cholangiocarcinomas

doi: 10.1016/j.ajpath.2015.02.010

Figure Lengend Snippet: Sequences of Primer Pairs (Sense and Antisense, Respectively) Used for Amplifying the Genes of Interest and the Internal Reference Gene ( GAPDH ) Used for Their Normalization

Article Snippet: For all immunoreactions, negative controls (the primary antibody was replaced with preimmune serum) were also included. shows the details of antibodies used in the study. table ft1 table-wrap mode="anchored" t5 caption a7 Name Host/isotype Source Catalog# Dilution CD326/EpCAM Mouse IgG1 Santa Cruz Biotechnology (Dallas, TX) sc-59782 1:50 K7 (cytokeratin 7) Mouse IgG1 Dako M7018 1:100 K19 (cytokeratin 19) Mouse IgG1 Abcam (Cambridge, UK) ab87014 1:50 K19 (cytokeratin 19) Mouse IgG1 Dako M0888 1:100 HepPar-1 Mouse IgG1 Dako M7158 1:50 CD133/Prominin 1 Rabbit IgG Abnova (Taipei, Taiwan) {"type":"entrez-protein","attrs":{"text":"PAB12663","term_id":"1236625334","term_text":"PAB12663"}} PAB12663 1:100 CD133/PROM1 Mouse IgG1 OriGene (Unimed Scientifica, Rome, Italy) TA309943 1:50 CD90/Thy1 Rabbit IgG Abcam ab92574 1:100 CD13 Mouse IgG1 Novacastra Reagents (Leica Biosystems, Buffalo Grove, IL) NCL-CD13-304 1:100 LGR5 Goat IgG Santa Cruz Biotechnology SC-68580 1:50 Desmin Mouse IgG1 Dako M0760 1:100 Vimentin Mouse IgG1 Santa Cruz Biotechnology sc-32322 1:100 Nestin Mouse IgG1 Santa Cruz Biotechnology sc-23927 1:100 α-SMA Mouse IgG1 Dako M0851 1:50 S100A4 Rabbit IgG Dako A5114 1:100 SNAIL Rabbit IgG Santa Cruz Biotechnology sc-28199 1:50 TWIST Rabbit IgG Santa Cruz Biotechnology sc-15393 1:50 LGR5 Rabbit IgG OriGene TA301323 1:50 NCAM-PE Mouse IgG1 BD Pharmingen (Milan, Italy) 555.516 1:50 E-cadherin Mouse IgG1 Santa Cruz Biotechnology sc-21791 1:50 P-cadherin Rabbit IgG Santa Cruz Biotechnology sc-7893 1:50 GFAP Mouse IgG1 Dako M0761 1:50 CD163 Mouse IgG1 OriGene TA506382 1:50 CD31 Mouse IgG1 Dako M0823 1:50 CD13 Mouse IgG1 Abcam Ab7417 1:100 SDF1 Rabbit IgG Santa Cruz Biotechnology sc-28876 1:50 FAP Mouse IgG1 Santa Cruz Biotechnology sc-65398 1:50 Periostin Goat IgG Santa Cruz Biotechnology sc-49480 1:50 Periostin Rabbit IgG Santa Cruz Biotechnology sc-67233 1:50 CD90-FITC Human Miltenyi Biotec (Cologne, Germany) 130-095-403 1:10 CD326 (EPCAM)-FITC Human Miltenyi Biotec 130-080-301 1:10 Goat anti-rabbit FITC IgG Abcam ab-6717 1:400 Goat anti-rabbit TRITC IgG Abcam ab-6718 1:400 Goat anti-mouse FITC IgG Abcam ab-6785 1:400 Goat anti-mouse TRITC IgG Abcam ab-6786 1:400 Open in a separate window List of Antibodies Used for IHC and IF Sections were examined in a coded fashion by Leica Microsystems DM 4500 B Light and Fluorescence Microscopy (Leica Microsystems, Weltzlar, Germany) equipped with a Jenoptik ProgRes C10 Plus Videocam (Jenoptik, Jena, Germany).

Techniques: Amplification

Journal: The American Journal of Pathology

Article Title: Profiles of Cancer Stem Cell Subpopulations in Cholangiocarcinomas

doi: 10.1016/j.ajpath.2015.02.010

Figure Lengend Snippet: Flow cytometry (FC) analyses of established cell lines of human cholangiocarcinomas (CCAs). A: Bar graphs showing the distribution, as percentages, of cancer stem cell (CSC) subpopulation in established human cancer cell lines as assessed by FC for CSC markers. B: FC representative plots. We investigated: i) HuH-28 and CCLP-I (mucin-negative) and HUCCT-1 (mucin-positive) cell lines derived from human IHCCA; ii) TFK-1 (mucin-positive) cell line derived from pCCA; and iii) Mz-ChA-1 (mucin-positive) cell line derived from gallbladder carcinoma. CD44 expression is high in most of the cell lines (>74%). CD90+ cells are evident primarily in HuH-28 and CCLP-1. EpCAM positivity characterizes the predominant cell population in the intrahepatic (IHCCA) cell line, HUCCT-1, in perihilar (pCCA) cell lines, TFK-1 and, in gallbladder cancer cell line, Mz-ChA-1. CD133 and LGR5 are minimally expressed. C: Bar graphs showing the distribution, as percentage, of CSC subpopulations coexpressing CSC markers. D: FC representative plots. CD44+/CD90+ cells predominate in HuH-28 and CCLP-1 cell lines. CD44+/EpCAM+ cells are prevalent in HUCCT-1, TFK-1, and Mz-ChA-1 cell lines. CD13+/CD90+ cells predominate only in HuH-28 cells. ∗∗P < 0.01 versus the other cell lines; ††P < 0.01 versus HuH28.

Article Snippet: For all immunoreactions, negative controls (the primary antibody was replaced with preimmune serum) were also included. shows the details of antibodies used in the study. table ft1 table-wrap mode="anchored" t5 caption a7 Name Host/isotype Source Catalog# Dilution CD326/EpCAM Mouse IgG1 Santa Cruz Biotechnology (Dallas, TX) sc-59782 1:50 K7 (cytokeratin 7) Mouse IgG1 Dako M7018 1:100 K19 (cytokeratin 19) Mouse IgG1 Abcam (Cambridge, UK) ab87014 1:50 K19 (cytokeratin 19) Mouse IgG1 Dako M0888 1:100 HepPar-1 Mouse IgG1 Dako M7158 1:50 CD133/Prominin 1 Rabbit IgG Abnova (Taipei, Taiwan) {"type":"entrez-protein","attrs":{"text":"PAB12663","term_id":"1236625334","term_text":"PAB12663"}} PAB12663 1:100 CD133/PROM1 Mouse IgG1 OriGene (Unimed Scientifica, Rome, Italy) TA309943 1:50 CD90/Thy1 Rabbit IgG Abcam ab92574 1:100 CD13 Mouse IgG1 Novacastra Reagents (Leica Biosystems, Buffalo Grove, IL) NCL-CD13-304 1:100 LGR5 Goat IgG Santa Cruz Biotechnology SC-68580 1:50 Desmin Mouse IgG1 Dako M0760 1:100 Vimentin Mouse IgG1 Santa Cruz Biotechnology sc-32322 1:100 Nestin Mouse IgG1 Santa Cruz Biotechnology sc-23927 1:100 α-SMA Mouse IgG1 Dako M0851 1:50 S100A4 Rabbit IgG Dako A5114 1:100 SNAIL Rabbit IgG Santa Cruz Biotechnology sc-28199 1:50 TWIST Rabbit IgG Santa Cruz Biotechnology sc-15393 1:50 LGR5 Rabbit IgG OriGene TA301323 1:50 NCAM-PE Mouse IgG1 BD Pharmingen (Milan, Italy) 555.516 1:50 E-cadherin Mouse IgG1 Santa Cruz Biotechnology sc-21791 1:50 P-cadherin Rabbit IgG Santa Cruz Biotechnology sc-7893 1:50 GFAP Mouse IgG1 Dako M0761 1:50 CD163 Mouse IgG1 OriGene TA506382 1:50 CD31 Mouse IgG1 Dako M0823 1:50 CD13 Mouse IgG1 Abcam Ab7417 1:100 SDF1 Rabbit IgG Santa Cruz Biotechnology sc-28876 1:50 FAP Mouse IgG1 Santa Cruz Biotechnology sc-65398 1:50 Periostin Goat IgG Santa Cruz Biotechnology sc-49480 1:50 Periostin Rabbit IgG Santa Cruz Biotechnology sc-67233 1:50 CD90-FITC Human Miltenyi Biotec (Cologne, Germany) 130-095-403 1:10 CD326 (EPCAM)-FITC Human Miltenyi Biotec 130-080-301 1:10 Goat anti-rabbit FITC IgG Abcam ab-6717 1:400 Goat anti-rabbit TRITC IgG Abcam ab-6718 1:400 Goat anti-mouse FITC IgG Abcam ab-6785 1:400 Goat anti-mouse TRITC IgG Abcam ab-6786 1:400 Open in a separate window List of Antibodies Used for IHC and IF Sections were examined in a coded fashion by Leica Microsystems DM 4500 B Light and Fluorescence Microscopy (Leica Microsystems, Weltzlar, Germany) equipped with a Jenoptik ProgRes C10 Plus Videocam (Jenoptik, Jena, Germany).

Techniques: Flow Cytometry, Derivative Assay, Expressing

Journal: The American Journal of Pathology

Article Title: Profiles of Cancer Stem Cell Subpopulations in Cholangiocarcinomas

doi: 10.1016/j.ajpath.2015.02.010

Figure Lengend Snippet: Immunophenotypic traits of human mucin-intrahepatic (IHCCA) and mixed-IHCCA. A: Periodic-acid Schiff (PAS) stain and immunohistochemistry (IHC) for keratin (K)7, EpCAM, LGR5, CD133, CD13, CD90, and α-smooth muscle actin (α-SMA) on human mucin-IHCCAs. Mucin-IHCCAs are diffusely positive for PAS, K7, EpCAM, LGR5, and CD133. By contrast, CD13 is restricted to a small subpopulation of tumor epithelial cells (arrow). CD90 and α-SMA are primarily expressed by tumor stromal cells (arrows). B: PAS stain and IHC for K7, EpCAM, LGR5, CD133, CD13, CD90, and α-SMA on human mixed-IHCCAs. Mixed-IHCCAs are diffusely positive for K7, EpCAM, CD13, and CD133. By contrast, LGR5 is restricted to a small subpopulation of tumor epithelial cells (arrows). CD90 and α-SMA were primarily expressed by tumor stromal cells (arrows). C: Double immunofluorescence (IF) for Desmin (red) and CD133 (green) in mucin-IHCCA; double IF for α-SMA (red) and pan-cytokeratin (Pan-CK; green) in mixed-IHCCAs. Approximately 10% of tumor epithelial cells (CD133+ or Pan-CK+, in green) express mesenchymal markers (desmin or α-SMA, in red) both in mucin- and in mixed-IHCCA. Yellow arrows indicate cells coexpressing epithelial and mesenchymal markers (yellow cells in merged images). Nuclei are shown in blue. Separate channels are shown. Boxed areas are shown magnified in the insets (bottom panels). D: Upper panels: IHC for SNAIL and Twist in human CCA specimens shows the expression of the two antigens within the nuclei of epithelial CCA cells (arrows). Lower panels: Double IF for SNAIL (red) and Pan-CK (green) in human CCA specimens confirms the expression of SNAIL within nuclei of epithelial CCA cells (arrows). Double IF for SNAIL (red) and α-SMA (green) in human CCA specimens shows the presence of CCA cells expressing both α-SMA and SNAIL. Images from mixed-IHCCA samples are displayed. E–J: IHC for CD133, CD13, and CD90 on human nontumor tissue surrounding CCA specimens. CD133 expression (E and F) is restricted to a few cells within Canals of Hering (CoH) and bile ductules (E, arrow) and to peribiliary glands (PBGs) within larger bile ducts (F, arrows). CD90 (G and H) is mostly expressed by stromal cells in the portal spaces (G, arrow) and around PBGs (H, arrows). CD13 (I and J) is diffusely expressed by hepatocytes, cells within CoH/bile ductules (I, yellow arrows), cholangiocytes of interlobular bile ducts (I, red arrow), and PBGs (J, arrow). Original magnification: ×20 (A–C, D, lower panels); ×40 (D, upper panel).

Article Snippet: For all immunoreactions, negative controls (the primary antibody was replaced with preimmune serum) were also included. shows the details of antibodies used in the study. table ft1 table-wrap mode="anchored" t5 caption a7 Name Host/isotype Source Catalog# Dilution CD326/EpCAM Mouse IgG1 Santa Cruz Biotechnology (Dallas, TX) sc-59782 1:50 K7 (cytokeratin 7) Mouse IgG1 Dako M7018 1:100 K19 (cytokeratin 19) Mouse IgG1 Abcam (Cambridge, UK) ab87014 1:50 K19 (cytokeratin 19) Mouse IgG1 Dako M0888 1:100 HepPar-1 Mouse IgG1 Dako M7158 1:50 CD133/Prominin 1 Rabbit IgG Abnova (Taipei, Taiwan) {"type":"entrez-protein","attrs":{"text":"PAB12663","term_id":"1236625334","term_text":"PAB12663"}} PAB12663 1:100 CD133/PROM1 Mouse IgG1 OriGene (Unimed Scientifica, Rome, Italy) TA309943 1:50 CD90/Thy1 Rabbit IgG Abcam ab92574 1:100 CD13 Mouse IgG1 Novacastra Reagents (Leica Biosystems, Buffalo Grove, IL) NCL-CD13-304 1:100 LGR5 Goat IgG Santa Cruz Biotechnology SC-68580 1:50 Desmin Mouse IgG1 Dako M0760 1:100 Vimentin Mouse IgG1 Santa Cruz Biotechnology sc-32322 1:100 Nestin Mouse IgG1 Santa Cruz Biotechnology sc-23927 1:100 α-SMA Mouse IgG1 Dako M0851 1:50 S100A4 Rabbit IgG Dako A5114 1:100 SNAIL Rabbit IgG Santa Cruz Biotechnology sc-28199 1:50 TWIST Rabbit IgG Santa Cruz Biotechnology sc-15393 1:50 LGR5 Rabbit IgG OriGene TA301323 1:50 NCAM-PE Mouse IgG1 BD Pharmingen (Milan, Italy) 555.516 1:50 E-cadherin Mouse IgG1 Santa Cruz Biotechnology sc-21791 1:50 P-cadherin Rabbit IgG Santa Cruz Biotechnology sc-7893 1:50 GFAP Mouse IgG1 Dako M0761 1:50 CD163 Mouse IgG1 OriGene TA506382 1:50 CD31 Mouse IgG1 Dako M0823 1:50 CD13 Mouse IgG1 Abcam Ab7417 1:100 SDF1 Rabbit IgG Santa Cruz Biotechnology sc-28876 1:50 FAP Mouse IgG1 Santa Cruz Biotechnology sc-65398 1:50 Periostin Goat IgG Santa Cruz Biotechnology sc-49480 1:50 Periostin Rabbit IgG Santa Cruz Biotechnology sc-67233 1:50 CD90-FITC Human Miltenyi Biotec (Cologne, Germany) 130-095-403 1:10 CD326 (EPCAM)-FITC Human Miltenyi Biotec 130-080-301 1:10 Goat anti-rabbit FITC IgG Abcam ab-6717 1:400 Goat anti-rabbit TRITC IgG Abcam ab-6718 1:400 Goat anti-mouse FITC IgG Abcam ab-6785 1:400 Goat anti-mouse TRITC IgG Abcam ab-6786 1:400 Open in a separate window List of Antibodies Used for IHC and IF Sections were examined in a coded fashion by Leica Microsystems DM 4500 B Light and Fluorescence Microscopy (Leica Microsystems, Weltzlar, Germany) equipped with a Jenoptik ProgRes C10 Plus Videocam (Jenoptik, Jena, Germany).

Techniques: Staining, Immunohistochemistry, Immunofluorescence, Expressing

Journal: The American Journal of Pathology

Article Title: Profiles of Cancer Stem Cell Subpopulations in Cholangiocarcinomas

doi: 10.1016/j.ajpath.2015.02.010

Figure Lengend Snippet: Human cholangiocarcinoma (CCA) primary cultures. A: Immunohistochemical analyses of CCA primary cultures after 2 to 3 passages (P2/P3) and 20 to 30 passages (P > 20). The expression of mesenchymal and epithelial-mesenchymal transition markers (vimentin, α-SMA, SNAIL, Twist, S100A4, P-cadherin) largely predominate over that of epithelial markers (CD133, EpCAM, LGR5, E-cadherin) in all passages examined. During progression of primary cultures (P > 20 versus P2/P3) SNAIL- and Twist-positive cells tend to increase, whereas EpCAM-, LGR5-, and CD133-positive cells tend to decrease. Similar diffuse positivity for vimentin, α-SMA, and S100A4 is observed in all passages. B–D: Flow cytometric (FC) analyses of primary cultures (20 to 30 passages) obtained from mixed-IHCCA (IH-mixed), mucin-IHCCA (IH-mucin), and mucin-pCCA (p-mucin) subtypes; representative FC plots. B: Cells positive for CD13, CD44, and CD90 largely predominate with respect to CD133, EpCAM, and LGR5. CD13+ and CD44+ cells predominate in mixed-IHCCA with respect to mucin-IHCCA or mucin-pCCA, whereas the opposite is found for CD90+ cells. C: Cells double-positive for CD13 and CD44 (CD13+/CD44+) predominate in mixed-IHCCA with respect to mucin-IHCCA or mucin-pCCA. D: CD90+/CD44−/CD13− cells predominate in mucin-IHCCAs and mucin-pCCA with respect to mixed-IHCCAs. In these experiments, CD90+ cells are gated and further analyzed for the expression of CD44 and CD13 markers. ∗P < 0.05, ∗∗P < 0.01 versus mucin-CCAs. Original magnification: ×40 (A).

Article Snippet: For all immunoreactions, negative controls (the primary antibody was replaced with preimmune serum) were also included. shows the details of antibodies used in the study. table ft1 table-wrap mode="anchored" t5 caption a7 Name Host/isotype Source Catalog# Dilution CD326/EpCAM Mouse IgG1 Santa Cruz Biotechnology (Dallas, TX) sc-59782 1:50 K7 (cytokeratin 7) Mouse IgG1 Dako M7018 1:100 K19 (cytokeratin 19) Mouse IgG1 Abcam (Cambridge, UK) ab87014 1:50 K19 (cytokeratin 19) Mouse IgG1 Dako M0888 1:100 HepPar-1 Mouse IgG1 Dako M7158 1:50 CD133/Prominin 1 Rabbit IgG Abnova (Taipei, Taiwan) {"type":"entrez-protein","attrs":{"text":"PAB12663","term_id":"1236625334","term_text":"PAB12663"}} PAB12663 1:100 CD133/PROM1 Mouse IgG1 OriGene (Unimed Scientifica, Rome, Italy) TA309943 1:50 CD90/Thy1 Rabbit IgG Abcam ab92574 1:100 CD13 Mouse IgG1 Novacastra Reagents (Leica Biosystems, Buffalo Grove, IL) NCL-CD13-304 1:100 LGR5 Goat IgG Santa Cruz Biotechnology SC-68580 1:50 Desmin Mouse IgG1 Dako M0760 1:100 Vimentin Mouse IgG1 Santa Cruz Biotechnology sc-32322 1:100 Nestin Mouse IgG1 Santa Cruz Biotechnology sc-23927 1:100 α-SMA Mouse IgG1 Dako M0851 1:50 S100A4 Rabbit IgG Dako A5114 1:100 SNAIL Rabbit IgG Santa Cruz Biotechnology sc-28199 1:50 TWIST Rabbit IgG Santa Cruz Biotechnology sc-15393 1:50 LGR5 Rabbit IgG OriGene TA301323 1:50 NCAM-PE Mouse IgG1 BD Pharmingen (Milan, Italy) 555.516 1:50 E-cadherin Mouse IgG1 Santa Cruz Biotechnology sc-21791 1:50 P-cadherin Rabbit IgG Santa Cruz Biotechnology sc-7893 1:50 GFAP Mouse IgG1 Dako M0761 1:50 CD163 Mouse IgG1 OriGene TA506382 1:50 CD31 Mouse IgG1 Dako M0823 1:50 CD13 Mouse IgG1 Abcam Ab7417 1:100 SDF1 Rabbit IgG Santa Cruz Biotechnology sc-28876 1:50 FAP Mouse IgG1 Santa Cruz Biotechnology sc-65398 1:50 Periostin Goat IgG Santa Cruz Biotechnology sc-49480 1:50 Periostin Rabbit IgG Santa Cruz Biotechnology sc-67233 1:50 CD90-FITC Human Miltenyi Biotec (Cologne, Germany) 130-095-403 1:10 CD326 (EPCAM)-FITC Human Miltenyi Biotec 130-080-301 1:10 Goat anti-rabbit FITC IgG Abcam ab-6717 1:400 Goat anti-rabbit TRITC IgG Abcam ab-6718 1:400 Goat anti-mouse FITC IgG Abcam ab-6785 1:400 Goat anti-mouse TRITC IgG Abcam ab-6786 1:400 Open in a separate window List of Antibodies Used for IHC and IF Sections were examined in a coded fashion by Leica Microsystems DM 4500 B Light and Fluorescence Microscopy (Leica Microsystems, Weltzlar, Germany) equipped with a Jenoptik ProgRes C10 Plus Videocam (Jenoptik, Jena, Germany).

Techniques: Immunohistochemical staining, Expressing

Journal: The American Journal of Pathology

Article Title: Profiles of Cancer Stem Cell Subpopulations in Cholangiocarcinomas

doi: 10.1016/j.ajpath.2015.02.010

Figure Lengend Snippet: Spheroids formed by cholangiocarcinomas (CCAs) subpopulations. A: We determined the capacity of spheroid formation by cancer stem cell (CSC) subpopulations immunosorted from CCA primary cultures; the number of spheroids (see bar graph) formed in vitro being a self-renewal index. By immunofluorescence (IF) for the marker used for immunosorting, we tested the purity of the formed spheroids. Spheroids formed by CD90+ cells are enriched for CD90+ cells, whereas the negative counterpart CD90− forms spheroids where CD90+ cells are <5%, indicating that these spheroids are formed by other CSC subpopulations. Each subpopulation forms spheroids efficiently, reaching a size of 100 to 500 μm after 7 days in culture. IF (nuclei were stained with DAPI) shows the enrichment in the spheroids by the immunosorted cell subpopulation. CD133, EpCAM, and LGR5 form a higher (P < 0.01) number of spheroids compared to cells expressing the mesenchymal cell marker, CD90, or CD13 (note that scales are different). CD13+ cells from mixed-IHCCA (IH-mixed) form a higher number of spheroids than CD13+ cells immunoselected from mucin-IHCCA (IH-mucin), whereas the opposite is observed for CD90+ or CD133+ cells (mucin > mixed.) B: Spheroids formed by CD133+ and CD90+ CSC subpopulations were analyzed by IF for markers of epithelial-mesenchymal transition (Twist, SNAIL, vimentin, P-cadherin). Spheroids show positive staining for Twist, SNAIL, vimentin, and P-cadherin without differences between CD133+ and CD90+ spheroids. ∗P < 0.05, ∗∗P < 0.01. Original magnification: ×30 (A); ×40 (B).

Article Snippet: For all immunoreactions, negative controls (the primary antibody was replaced with preimmune serum) were also included. shows the details of antibodies used in the study. table ft1 table-wrap mode="anchored" t5 caption a7 Name Host/isotype Source Catalog# Dilution CD326/EpCAM Mouse IgG1 Santa Cruz Biotechnology (Dallas, TX) sc-59782 1:50 K7 (cytokeratin 7) Mouse IgG1 Dako M7018 1:100 K19 (cytokeratin 19) Mouse IgG1 Abcam (Cambridge, UK) ab87014 1:50 K19 (cytokeratin 19) Mouse IgG1 Dako M0888 1:100 HepPar-1 Mouse IgG1 Dako M7158 1:50 CD133/Prominin 1 Rabbit IgG Abnova (Taipei, Taiwan) {"type":"entrez-protein","attrs":{"text":"PAB12663","term_id":"1236625334","term_text":"PAB12663"}} PAB12663 1:100 CD133/PROM1 Mouse IgG1 OriGene (Unimed Scientifica, Rome, Italy) TA309943 1:50 CD90/Thy1 Rabbit IgG Abcam ab92574 1:100 CD13 Mouse IgG1 Novacastra Reagents (Leica Biosystems, Buffalo Grove, IL) NCL-CD13-304 1:100 LGR5 Goat IgG Santa Cruz Biotechnology SC-68580 1:50 Desmin Mouse IgG1 Dako M0760 1:100 Vimentin Mouse IgG1 Santa Cruz Biotechnology sc-32322 1:100 Nestin Mouse IgG1 Santa Cruz Biotechnology sc-23927 1:100 α-SMA Mouse IgG1 Dako M0851 1:50 S100A4 Rabbit IgG Dako A5114 1:100 SNAIL Rabbit IgG Santa Cruz Biotechnology sc-28199 1:50 TWIST Rabbit IgG Santa Cruz Biotechnology sc-15393 1:50 LGR5 Rabbit IgG OriGene TA301323 1:50 NCAM-PE Mouse IgG1 BD Pharmingen (Milan, Italy) 555.516 1:50 E-cadherin Mouse IgG1 Santa Cruz Biotechnology sc-21791 1:50 P-cadherin Rabbit IgG Santa Cruz Biotechnology sc-7893 1:50 GFAP Mouse IgG1 Dako M0761 1:50 CD163 Mouse IgG1 OriGene TA506382 1:50 CD31 Mouse IgG1 Dako M0823 1:50 CD13 Mouse IgG1 Abcam Ab7417 1:100 SDF1 Rabbit IgG Santa Cruz Biotechnology sc-28876 1:50 FAP Mouse IgG1 Santa Cruz Biotechnology sc-65398 1:50 Periostin Goat IgG Santa Cruz Biotechnology sc-49480 1:50 Periostin Rabbit IgG Santa Cruz Biotechnology sc-67233 1:50 CD90-FITC Human Miltenyi Biotec (Cologne, Germany) 130-095-403 1:10 CD326 (EPCAM)-FITC Human Miltenyi Biotec 130-080-301 1:10 Goat anti-rabbit FITC IgG Abcam ab-6717 1:400 Goat anti-rabbit TRITC IgG Abcam ab-6718 1:400 Goat anti-mouse FITC IgG Abcam ab-6785 1:400 Goat anti-mouse TRITC IgG Abcam ab-6786 1:400 Open in a separate window List of Antibodies Used for IHC and IF Sections were examined in a coded fashion by Leica Microsystems DM 4500 B Light and Fluorescence Microscopy (Leica Microsystems, Weltzlar, Germany) equipped with a Jenoptik ProgRes C10 Plus Videocam (Jenoptik, Jena, Germany).

Techniques: In Vitro, Immunofluorescence, Marker, Staining, Expressing

Journal: The American Journal of Pathology

Article Title: Profiles of Cancer Stem Cell Subpopulations in Cholangiocarcinomas

doi: 10.1016/j.ajpath.2015.02.010

Figure Lengend Snippet: Tumor Xenografts Obtained 2 Months after S.C. Injection of Spheroids Prepared from Different Subpopulations of Immunosorted CSCs

Article Snippet: For all immunoreactions, negative controls (the primary antibody was replaced with preimmune serum) were also included. shows the details of antibodies used in the study. table ft1 table-wrap mode="anchored" t5 caption a7 Name Host/isotype Source Catalog# Dilution CD326/EpCAM Mouse IgG1 Santa Cruz Biotechnology (Dallas, TX) sc-59782 1:50 K7 (cytokeratin 7) Mouse IgG1 Dako M7018 1:100 K19 (cytokeratin 19) Mouse IgG1 Abcam (Cambridge, UK) ab87014 1:50 K19 (cytokeratin 19) Mouse IgG1 Dako M0888 1:100 HepPar-1 Mouse IgG1 Dako M7158 1:50 CD133/Prominin 1 Rabbit IgG Abnova (Taipei, Taiwan) {"type":"entrez-protein","attrs":{"text":"PAB12663","term_id":"1236625334","term_text":"PAB12663"}} PAB12663 1:100 CD133/PROM1 Mouse IgG1 OriGene (Unimed Scientifica, Rome, Italy) TA309943 1:50 CD90/Thy1 Rabbit IgG Abcam ab92574 1:100 CD13 Mouse IgG1 Novacastra Reagents (Leica Biosystems, Buffalo Grove, IL) NCL-CD13-304 1:100 LGR5 Goat IgG Santa Cruz Biotechnology SC-68580 1:50 Desmin Mouse IgG1 Dako M0760 1:100 Vimentin Mouse IgG1 Santa Cruz Biotechnology sc-32322 1:100 Nestin Mouse IgG1 Santa Cruz Biotechnology sc-23927 1:100 α-SMA Mouse IgG1 Dako M0851 1:50 S100A4 Rabbit IgG Dako A5114 1:100 SNAIL Rabbit IgG Santa Cruz Biotechnology sc-28199 1:50 TWIST Rabbit IgG Santa Cruz Biotechnology sc-15393 1:50 LGR5 Rabbit IgG OriGene TA301323 1:50 NCAM-PE Mouse IgG1 BD Pharmingen (Milan, Italy) 555.516 1:50 E-cadherin Mouse IgG1 Santa Cruz Biotechnology sc-21791 1:50 P-cadherin Rabbit IgG Santa Cruz Biotechnology sc-7893 1:50 GFAP Mouse IgG1 Dako M0761 1:50 CD163 Mouse IgG1 OriGene TA506382 1:50 CD31 Mouse IgG1 Dako M0823 1:50 CD13 Mouse IgG1 Abcam Ab7417 1:100 SDF1 Rabbit IgG Santa Cruz Biotechnology sc-28876 1:50 FAP Mouse IgG1 Santa Cruz Biotechnology sc-65398 1:50 Periostin Goat IgG Santa Cruz Biotechnology sc-49480 1:50 Periostin Rabbit IgG Santa Cruz Biotechnology sc-67233 1:50 CD90-FITC Human Miltenyi Biotec (Cologne, Germany) 130-095-403 1:10 CD326 (EPCAM)-FITC Human Miltenyi Biotec 130-080-301 1:10 Goat anti-rabbit FITC IgG Abcam ab-6717 1:400 Goat anti-rabbit TRITC IgG Abcam ab-6718 1:400 Goat anti-mouse FITC IgG Abcam ab-6785 1:400 Goat anti-mouse TRITC IgG Abcam ab-6786 1:400 Open in a separate window List of Antibodies Used for IHC and IF Sections were examined in a coded fashion by Leica Microsystems DM 4500 B Light and Fluorescence Microscopy (Leica Microsystems, Weltzlar, Germany) equipped with a Jenoptik ProgRes C10 Plus Videocam (Jenoptik, Jena, Germany).

Techniques: Injection

Journal: The American Journal of Pathology

Article Title: Profiles of Cancer Stem Cell Subpopulations in Cholangiocarcinomas

doi: 10.1016/j.ajpath.2015.02.010

Figure Lengend Snippet: Histology of s.c. human tumor xenografts. A: Hematoxylin and eosin (H&E): Xenografts arising from CD90+, EpCAM+, and LGR5+ spheroids are characterized by the presence of larger necrotic areas in comparison with tumors obtained from CD133+ cells, whereas in xenografts from CD13+ spheroids, only few and restricted necrotic areas are observed. Necrotic areas are encircled by the dotted line. B: H&E: All s.c. xenografts comprise of nests of pleomorphic cells with giant and irregular nuclei and multiple prominent nucleoli. Numerous tumor cells are PCNA positive. Most tumor cells are α-SMA positive (arrows), whereas few and scattered nests of tumor cells express K19 (arrows). C: Spheroids from mucin-IHCCA, injected s.c., give rise to xenografts in which neoplastic cells are surrounded by a Periodic-acid Schiff (PAS)-positive material (arrows). D: EpCAM+ and LGR5+ spheroids from mucin-IHCCA show the presence of few areas composed of K19+ duct-like structures (arrows). E: Spheroids from mixed-IHCCA give rise to xenografts that are almost PAS negative. F: 5% to 30% of cells are K19 positive and form few ductular-like structures (arrows). Original magnification: ×10 (A); ×40 (B–F).

Article Snippet: For all immunoreactions, negative controls (the primary antibody was replaced with preimmune serum) were also included. shows the details of antibodies used in the study. table ft1 table-wrap mode="anchored" t5 caption a7 Name Host/isotype Source Catalog# Dilution CD326/EpCAM Mouse IgG1 Santa Cruz Biotechnology (Dallas, TX) sc-59782 1:50 K7 (cytokeratin 7) Mouse IgG1 Dako M7018 1:100 K19 (cytokeratin 19) Mouse IgG1 Abcam (Cambridge, UK) ab87014 1:50 K19 (cytokeratin 19) Mouse IgG1 Dako M0888 1:100 HepPar-1 Mouse IgG1 Dako M7158 1:50 CD133/Prominin 1 Rabbit IgG Abnova (Taipei, Taiwan) {"type":"entrez-protein","attrs":{"text":"PAB12663","term_id":"1236625334","term_text":"PAB12663"}} PAB12663 1:100 CD133/PROM1 Mouse IgG1 OriGene (Unimed Scientifica, Rome, Italy) TA309943 1:50 CD90/Thy1 Rabbit IgG Abcam ab92574 1:100 CD13 Mouse IgG1 Novacastra Reagents (Leica Biosystems, Buffalo Grove, IL) NCL-CD13-304 1:100 LGR5 Goat IgG Santa Cruz Biotechnology SC-68580 1:50 Desmin Mouse IgG1 Dako M0760 1:100 Vimentin Mouse IgG1 Santa Cruz Biotechnology sc-32322 1:100 Nestin Mouse IgG1 Santa Cruz Biotechnology sc-23927 1:100 α-SMA Mouse IgG1 Dako M0851 1:50 S100A4 Rabbit IgG Dako A5114 1:100 SNAIL Rabbit IgG Santa Cruz Biotechnology sc-28199 1:50 TWIST Rabbit IgG Santa Cruz Biotechnology sc-15393 1:50 LGR5 Rabbit IgG OriGene TA301323 1:50 NCAM-PE Mouse IgG1 BD Pharmingen (Milan, Italy) 555.516 1:50 E-cadherin Mouse IgG1 Santa Cruz Biotechnology sc-21791 1:50 P-cadherin Rabbit IgG Santa Cruz Biotechnology sc-7893 1:50 GFAP Mouse IgG1 Dako M0761 1:50 CD163 Mouse IgG1 OriGene TA506382 1:50 CD31 Mouse IgG1 Dako M0823 1:50 CD13 Mouse IgG1 Abcam Ab7417 1:100 SDF1 Rabbit IgG Santa Cruz Biotechnology sc-28876 1:50 FAP Mouse IgG1 Santa Cruz Biotechnology sc-65398 1:50 Periostin Goat IgG Santa Cruz Biotechnology sc-49480 1:50 Periostin Rabbit IgG Santa Cruz Biotechnology sc-67233 1:50 CD90-FITC Human Miltenyi Biotec (Cologne, Germany) 130-095-403 1:10 CD326 (EPCAM)-FITC Human Miltenyi Biotec 130-080-301 1:10 Goat anti-rabbit FITC IgG Abcam ab-6717 1:400 Goat anti-rabbit TRITC IgG Abcam ab-6718 1:400 Goat anti-mouse FITC IgG Abcam ab-6785 1:400 Goat anti-mouse TRITC IgG Abcam ab-6786 1:400 Open in a separate window List of Antibodies Used for IHC and IF Sections were examined in a coded fashion by Leica Microsystems DM 4500 B Light and Fluorescence Microscopy (Leica Microsystems, Weltzlar, Germany) equipped with a Jenoptik ProgRes C10 Plus Videocam (Jenoptik, Jena, Germany).

Techniques: Injection

Journal: The American Journal of Pathology

Article Title: Profiles of Cancer Stem Cell Subpopulations in Cholangiocarcinomas

doi: 10.1016/j.ajpath.2015.02.010

Figure Lengend Snippet: Summary of Morphological and IHC Features of S.C. Xenografts ∗

Article Snippet: For all immunoreactions, negative controls (the primary antibody was replaced with preimmune serum) were also included. shows the details of antibodies used in the study. table ft1 table-wrap mode="anchored" t5 caption a7 Name Host/isotype Source Catalog# Dilution CD326/EpCAM Mouse IgG1 Santa Cruz Biotechnology (Dallas, TX) sc-59782 1:50 K7 (cytokeratin 7) Mouse IgG1 Dako M7018 1:100 K19 (cytokeratin 19) Mouse IgG1 Abcam (Cambridge, UK) ab87014 1:50 K19 (cytokeratin 19) Mouse IgG1 Dako M0888 1:100 HepPar-1 Mouse IgG1 Dako M7158 1:50 CD133/Prominin 1 Rabbit IgG Abnova (Taipei, Taiwan) {"type":"entrez-protein","attrs":{"text":"PAB12663","term_id":"1236625334","term_text":"PAB12663"}} PAB12663 1:100 CD133/PROM1 Mouse IgG1 OriGene (Unimed Scientifica, Rome, Italy) TA309943 1:50 CD90/Thy1 Rabbit IgG Abcam ab92574 1:100 CD13 Mouse IgG1 Novacastra Reagents (Leica Biosystems, Buffalo Grove, IL) NCL-CD13-304 1:100 LGR5 Goat IgG Santa Cruz Biotechnology SC-68580 1:50 Desmin Mouse IgG1 Dako M0760 1:100 Vimentin Mouse IgG1 Santa Cruz Biotechnology sc-32322 1:100 Nestin Mouse IgG1 Santa Cruz Biotechnology sc-23927 1:100 α-SMA Mouse IgG1 Dako M0851 1:50 S100A4 Rabbit IgG Dako A5114 1:100 SNAIL Rabbit IgG Santa Cruz Biotechnology sc-28199 1:50 TWIST Rabbit IgG Santa Cruz Biotechnology sc-15393 1:50 LGR5 Rabbit IgG OriGene TA301323 1:50 NCAM-PE Mouse IgG1 BD Pharmingen (Milan, Italy) 555.516 1:50 E-cadherin Mouse IgG1 Santa Cruz Biotechnology sc-21791 1:50 P-cadherin Rabbit IgG Santa Cruz Biotechnology sc-7893 1:50 GFAP Mouse IgG1 Dako M0761 1:50 CD163 Mouse IgG1 OriGene TA506382 1:50 CD31 Mouse IgG1 Dako M0823 1:50 CD13 Mouse IgG1 Abcam Ab7417 1:100 SDF1 Rabbit IgG Santa Cruz Biotechnology sc-28876 1:50 FAP Mouse IgG1 Santa Cruz Biotechnology sc-65398 1:50 Periostin Goat IgG Santa Cruz Biotechnology sc-49480 1:50 Periostin Rabbit IgG Santa Cruz Biotechnology sc-67233 1:50 CD90-FITC Human Miltenyi Biotec (Cologne, Germany) 130-095-403 1:10 CD326 (EPCAM)-FITC Human Miltenyi Biotec 130-080-301 1:10 Goat anti-rabbit FITC IgG Abcam ab-6717 1:400 Goat anti-rabbit TRITC IgG Abcam ab-6718 1:400 Goat anti-mouse FITC IgG Abcam ab-6785 1:400 Goat anti-mouse TRITC IgG Abcam ab-6786 1:400 Open in a separate window List of Antibodies Used for IHC and IF Sections were examined in a coded fashion by Leica Microsystems DM 4500 B Light and Fluorescence Microscopy (Leica Microsystems, Weltzlar, Germany) equipped with a Jenoptik ProgRes C10 Plus Videocam (Jenoptik, Jena, Germany).

Techniques:

Journal: The American Journal of Pathology

Article Title: Profiles of Cancer Stem Cell Subpopulations in Cholangiocarcinomas

doi: 10.1016/j.ajpath.2015.02.010

Figure Lengend Snippet: Intrahepatic tumor xenografts: morphological and phenotypic features. Injection of spheroids (approximately 10,000 cells) formed by cells immunoselected for a determined cholangiocarcinoma (CCA) marker into the livers of cirrhotic SCID mice (carbon tetrachloride–induced) leads, after 4 weeks, to evident liver cancers. A: Tumor masses observed 4 weeks after intrahepatic injection of CD90+ spheroids from primary cultures of mucin-IHCCA. B: Hematoxylin and eosin (H&E): The liver is occupied by several tumor masses (dotted line); vascular invasion (arrows). C: H&E: Tumor masses were composed of strands of polygonal cells with giant nuclei and prominent nucleoli. D: Immunohistochemistry (IHC): At the center of tumor masses, α-SMA–positive tumor cells are present (arrows). E–H: At the periphery of the tumor masses, Periodic-acid Schiff (PAS)-positive cells (F, arrows), cords of HepPar-1–positive cells (G, arrows), and K19 ductular-like structures (H, arrows) are present, reproducing a moderately differentiated carcinoma. E = PAS staining: Area in the box is magnified in F; F = PAS staining: G = IHC for HepPar-1. H = IHC for CK19. I and J: Immunocompetent cirrhotic BALBc mice, under pharmacological immunosuppression, injected with CD133+ spheroids prepared from mucin-IHCCA primary cultures. Four weeks after intrahepatic injection, a tumor almost totally composed of PAS+ duct-like structures (arrows) is observed. I = H&E: J = PAS staining. n = 4 (I and J, mucin-IHCCA primary cultures). Original magnification: ×10 (B and E); ×20 (C, D, H, and I); ×40 (F, G, and J).

Article Snippet: For all immunoreactions, negative controls (the primary antibody was replaced with preimmune serum) were also included. shows the details of antibodies used in the study. table ft1 table-wrap mode="anchored" t5 caption a7 Name Host/isotype Source Catalog# Dilution CD326/EpCAM Mouse IgG1 Santa Cruz Biotechnology (Dallas, TX) sc-59782 1:50 K7 (cytokeratin 7) Mouse IgG1 Dako M7018 1:100 K19 (cytokeratin 19) Mouse IgG1 Abcam (Cambridge, UK) ab87014 1:50 K19 (cytokeratin 19) Mouse IgG1 Dako M0888 1:100 HepPar-1 Mouse IgG1 Dako M7158 1:50 CD133/Prominin 1 Rabbit IgG Abnova (Taipei, Taiwan) {"type":"entrez-protein","attrs":{"text":"PAB12663","term_id":"1236625334","term_text":"PAB12663"}} PAB12663 1:100 CD133/PROM1 Mouse IgG1 OriGene (Unimed Scientifica, Rome, Italy) TA309943 1:50 CD90/Thy1 Rabbit IgG Abcam ab92574 1:100 CD13 Mouse IgG1 Novacastra Reagents (Leica Biosystems, Buffalo Grove, IL) NCL-CD13-304 1:100 LGR5 Goat IgG Santa Cruz Biotechnology SC-68580 1:50 Desmin Mouse IgG1 Dako M0760 1:100 Vimentin Mouse IgG1 Santa Cruz Biotechnology sc-32322 1:100 Nestin Mouse IgG1 Santa Cruz Biotechnology sc-23927 1:100 α-SMA Mouse IgG1 Dako M0851 1:50 S100A4 Rabbit IgG Dako A5114 1:100 SNAIL Rabbit IgG Santa Cruz Biotechnology sc-28199 1:50 TWIST Rabbit IgG Santa Cruz Biotechnology sc-15393 1:50 LGR5 Rabbit IgG OriGene TA301323 1:50 NCAM-PE Mouse IgG1 BD Pharmingen (Milan, Italy) 555.516 1:50 E-cadherin Mouse IgG1 Santa Cruz Biotechnology sc-21791 1:50 P-cadherin Rabbit IgG Santa Cruz Biotechnology sc-7893 1:50 GFAP Mouse IgG1 Dako M0761 1:50 CD163 Mouse IgG1 OriGene TA506382 1:50 CD31 Mouse IgG1 Dako M0823 1:50 CD13 Mouse IgG1 Abcam Ab7417 1:100 SDF1 Rabbit IgG Santa Cruz Biotechnology sc-28876 1:50 FAP Mouse IgG1 Santa Cruz Biotechnology sc-65398 1:50 Periostin Goat IgG Santa Cruz Biotechnology sc-49480 1:50 Periostin Rabbit IgG Santa Cruz Biotechnology sc-67233 1:50 CD90-FITC Human Miltenyi Biotec (Cologne, Germany) 130-095-403 1:10 CD326 (EPCAM)-FITC Human Miltenyi Biotec 130-080-301 1:10 Goat anti-rabbit FITC IgG Abcam ab-6717 1:400 Goat anti-rabbit TRITC IgG Abcam ab-6718 1:400 Goat anti-mouse FITC IgG Abcam ab-6785 1:400 Goat anti-mouse TRITC IgG Abcam ab-6786 1:400 Open in a separate window List of Antibodies Used for IHC and IF Sections were examined in a coded fashion by Leica Microsystems DM 4500 B Light and Fluorescence Microscopy (Leica Microsystems, Weltzlar, Germany) equipped with a Jenoptik ProgRes C10 Plus Videocam (Jenoptik, Jena, Germany).

Techniques: Injection, Marker, Immunohistochemistry, Staining

Journal: British Journal of Cancer

Article Title: CD133/prominin-1 is a potential therapeutic target for antibody-drug conjugates in hepatocellular and gastric cancers

doi: 10.1038/sj.bjc.6604437

Figure Lengend Snippet: Immunohistochemical analysis of CD133 expression demonstrating the scoring intensity and expression pattern. Representative images using anti-CD133 MAb ab5558: ( A ) liver cholangiocarcinoma with multifocal, minimal to mild membranous and cytoplasmic staining, ( B ) pancreatic adenocarcinoma with mild to moderate membranous (apical) staining of luminal structures, ( C ) gastric adenocarcinoma with moderate to strong staining in two distinct cell populations: (1) luminal and apical and (2) cytoplasmic and membranous, ( D ) normal liver with minimal and nonspecific cytoplasmic staining of hepatocytes and apical staining of bile duct, ( E ) normal pancreas with weak to mild, apical membranous staining of acinar epithelium and ductal epithelium, ( F ) normal stomach with minimal to mild apical staining of glandular crypt epithelium. The scale bars represent 50 μ m.

Article Snippet: For mouse xenograft tumours, a rabbit anti-CD133 MAb (Cell Signaling Technology, Danvers, MA, USA) and a rabbit polyclonal anti-CD133 (ab19898, Abcam) were used separately as primary antibodies and the Bond Polymer Alkaline Phosphate (AP) Red Detection kit (Vision BioSystems, Australia) for detection.

Techniques: Immunohistochemical staining, Expressing, Staining

Journal: British Journal of Cancer

Article Title: CD133/prominin-1 is a potential therapeutic target for antibody-drug conjugates in hepatocellular and gastric cancers

doi: 10.1038/sj.bjc.6604437

Figure Lengend Snippet: CD133 expression analysis by immunohistochemistry

Article Snippet: For mouse xenograft tumours, a rabbit anti-CD133 MAb (Cell Signaling Technology, Danvers, MA, USA) and a rabbit polyclonal anti-CD133 (ab19898, Abcam) were used separately as primary antibodies and the Bond Polymer Alkaline Phosphate (AP) Red Detection kit (Vision BioSystems, Australia) for detection.

Techniques: Expressing, Immunohistochemistry

Journal: British Journal of Cancer

Article Title: CD133/prominin-1 is a potential therapeutic target for antibody-drug conjugates in hepatocellular and gastric cancers

doi: 10.1038/sj.bjc.6604437

Figure Lengend Snippet: Tumour and normal cell line expression of CD133 and sensitivity to anti-CD133 antibody-drug conjugate, AC133-vcMMAF

Article Snippet: For mouse xenograft tumours, a rabbit anti-CD133 MAb (Cell Signaling Technology, Danvers, MA, USA) and a rabbit polyclonal anti-CD133 (ab19898, Abcam) were used separately as primary antibodies and the Bond Polymer Alkaline Phosphate (AP) Red Detection kit (Vision BioSystems, Australia) for detection.

Techniques: Expressing, Inhibition

Journal: British Journal of Cancer

Article Title: CD133/prominin-1 is a potential therapeutic target for antibody-drug conjugates in hepatocellular and gastric cancers

doi: 10.1038/sj.bjc.6604437

Figure Lengend Snippet: Activity of anti-CD133 ADC against cancer cell lines. ( A ) ADCs targeting CD133 have potent cytotoxic activity against antigen-positive hepatocellular and gastric carcinoma cell lines. Cytotoxicity was measured by resazurin dye conversion in Hep3B and KATO III cells grown in 96-well plates and exposed to anti-CD133 (AC133-vcMMAF) and control ADCs (IgG-vcMMAF and OKT9-vcMMAF) and crosslinked unconjugated anti-CD133 MAb (AC133) for 96 h. ( B ) Proliferation was measured using [ 3 H]-thymidine uptake in Hep3B and KATO III cells grown in 96-well plates and exposed to anti-CD133 and control ADCs for 96 h. ( C ) Induction of apoptosis in Hep3B cells treated with AC133-vcMMAF. Caspase 3/7 activation, a quantitative measurement of apoptotic cells, was monitored using the Caspase Glo assay at various time points (24–72 h) after addition of ADCs. Caspase 3/7 activation relative to untreated cells was detected by 48 h with optimal measurement after 72 h in Hep3B cells treated with increasing concentrations of AC133-vcMMAF and positive control OKT9-vcMMAF. ( D ) Inhibition of ADC cytotoxicity using internalisation inhibitor, ammonium chloride (NH 4 Cl) in Hep3B cells. Cells were incubated with increasing concentration of NH 4 Cl 30 min before treated with anti-CD133 (AC133-vcMMAF) or control ADCs. Cytotoxicity was measured after 72 h using the rezasurin dye conversion as in ( A ). The percentage inhibition of cytotoxicity relative to control untreated cells at an ADC concentration of 400 ng ml −1 is shown.

Article Snippet: For mouse xenograft tumours, a rabbit anti-CD133 MAb (Cell Signaling Technology, Danvers, MA, USA) and a rabbit polyclonal anti-CD133 (ab19898, Abcam) were used separately as primary antibodies and the Bond Polymer Alkaline Phosphate (AP) Red Detection kit (Vision BioSystems, Australia) for detection.

Techniques: Activity Assay, Control, Activation Assay, Caspase-Glo Assay, Positive Control, Inhibition, Incubation, Concentration Assay

Journal: British Journal of Cancer

Article Title: CD133/prominin-1 is a potential therapeutic target for antibody-drug conjugates in hepatocellular and gastric cancers

doi: 10.1038/sj.bjc.6604437

Figure Lengend Snippet: Subcellular localisation of anti-CD133 ADC, AC133-vcMMAF, in sensitive and resistant cancer cell lines. ( A ) AC133-vcMMAF, partially colocalises (yellow) with the lysosomal marker, CD107a, in Hep3B and KATO III cells. Subcellular localisation of AC133-vcMMAF (red) and CD107a (green) in Hep3B and KATO III cells after 24 h incubation with the ADC. ( B ) Subcellular localisation of AC133-vcMMAF, lysosomal marker, CD107a, and caveolin-1 (Cav-1) in Su.86.86 after 24 h incubation with the ADC. AC133-vcMMAF colocalises with Cav-1 (yellow) and not with CD107a in this resistant cell line. Nuclei were stained blue with DAPI. Images were acquired using a × 63 oil immersion objective with Apotome for optical sectioning.

Article Snippet: For mouse xenograft tumours, a rabbit anti-CD133 MAb (Cell Signaling Technology, Danvers, MA, USA) and a rabbit polyclonal anti-CD133 (ab19898, Abcam) were used separately as primary antibodies and the Bond Polymer Alkaline Phosphate (AP) Red Detection kit (Vision BioSystems, Australia) for detection.

Techniques: Marker, Incubation, Staining

Journal: British Journal of Cancer

Article Title: CD133/prominin-1 is a potential therapeutic target for antibody-drug conjugates in hepatocellular and gastric cancers

doi: 10.1038/sj.bjc.6604437

Figure Lengend Snippet: In vivo efficacy of an anti-CD133 ADC in Hep3B hepatocellular carcinoma model including IHC analysis of CD133 expression following ADC treatment. ( A ) In vivo efficacy of AC133-vcMMAF in Hep3B subcutaneous tumours. SCID mice ( n =7/group) with established (∼100 mm 3 ) Hep3B tumour xenografts were treated by intraperitoneal injection every 4 days for a total of four doses (red arrows) with the anti-CD133 antibody (AC133) or ADC (AC133-vcMMAF) or isotype control mouse IgG1-vcMMAF. An additional group of mice was left untreated as a control. Median tumour volume plots were continued for each group until one or more animals died or were euthanised (see Materials and methods). Tumours were collected when the tumour volume reached 1000 mm 3 . Highly concordant data were in an independent replicate of this experiment. ( B ) CD133 expression in Hep3B xenograft tumours after anti-CD133 drug conjugate treatment. IHC analysis using rabbit anti-CD133 MAb in ( a ) untreated Hep3B xenograft, ( b ) treatment with control IgG-vcMMAF (3.0 mg kg −1 ) and ( c ) treated with AC133-vcMMAF (3.0 mg kg −1 ). ( d ) same tumour as ( c ) stained with control rabbit IgG as primary antibody. Fast Red chromagen was used to detect CD133 expression.

Article Snippet: For mouse xenograft tumours, a rabbit anti-CD133 MAb (Cell Signaling Technology, Danvers, MA, USA) and a rabbit polyclonal anti-CD133 (ab19898, Abcam) were used separately as primary antibodies and the Bond Polymer Alkaline Phosphate (AP) Red Detection kit (Vision BioSystems, Australia) for detection.

Techniques: In Vivo, Expressing, Injection, Control, Staining