Journal: The American Journal of Pathology

Article Title: Transforming Growth Factors α and β Are Essential for Modeling Cholangiocarcinoma Desmoplasia and Progression in a Three-Dimensional Organotypic Culture Model

doi: 10.1016/j.ajpath.2017.01.013

Figure Lengend Snippet: Differential immunophenotyping by biomarker immunofluorescence staining of TDECC cholangiocarcinoma versus TDFSM cell strains individually cultured for 24 to 48 hours on rat tail collagen type I–coated glass coverslips in standard medium containing 10% FBS. Primary antibody dilutions are shown in parentheses. In each case, corresponding controls with secondary antibody alone, omitting the primary antibody, were negative for immunofluorescence staining. Green staining: secondary antibody conjugated with Alexa Fluor 488; orange staining: secondary antibody conjugated with Alexa Fluor 546; blue nuclei: DAPI staining. Scale bars: 50 μm (except for NTPDase 2); 40 μm (NTPDase 2). α-SMA, α-smooth muscle actin; CK19, cytokeratin-19; NTPDase 2, nucleoside triphosphate diphosphohydrolase 2; Postn, periostin; Thy-1, thymocyte differentiation antigen-1; Vim, vimentin.

Article Snippet: 26 , 27 , 28 Immunofluorescence biomarker profile staining of the individual cholangiocarcinoma and fibroblastic cell strains cultured on collagen type I–coated coverslips and fixed for approximately 10 minutes with ice-cold 4% paraformaldehyde in phosphate-buffered saline, pH 7.4, was performed using the following primary antibodies: CK19 mouse monoclonal (VP-C415, b170) (Vector Laboratories, Inc., Burlingame, CA), α-SMA mouse monoclonal (M0851, 1A4) (Dako Cytomation, Inc., Carpinteria, CA), desmin polyclonal goat IgG (sc-7559, Y-20), vimentin (Vim) mouse monoclonal (sc-6260, V9), thymocyte differentiation antigen-1 (Thy-1) mouse monoclonal (sc-53116, OX7), CD34 mouse monoclonal (sc-7324, ICO115), and CD45 mouse monoclonal (sc-53047, OX30) (Santa Cruz Biotechnology, Inc., Paso Robles, CA), anti-rat elastin rabbit polyclonal IgG (CL55041AP, Lot 333K) (Cedarlane Labs, Burlington, NC), and anti-Postn rabbit polyclonal IgG (ab14041) (Abcam, Inc., Cambridge, MA).

Techniques: Biomarker Assay, Immunofluorescence, Staining, Cell Culture

Journal: The American Journal of Pathology

Article Title: Transforming Growth Factors α and β Are Essential for Modeling Cholangiocarcinoma Desmoplasia and Progression in a Three-Dimensional Organotypic Culture Model

doi: 10.1016/j.ajpath.2017.01.013

Figure Lengend Snippet: A: Quantitative Western blot analysis of select protein biomarkers differentially expressed in cultured TDFSM and TDECC cell strains individually grown to 95% to 100% confluency under standard culture conditions (10% FBS supplemented medium) in 10-cm-diameter plastic culture dishes coated with rat tail collagen type I. B: In the case of the secreted protein Postn, Western blotting was performed on concentrated medium samples prepared using an Amicon Ultra-2 Centrifugal Filter Device with Ultracel-50 membrane (catalog number UFC205024; EMD Millipore Corp., Darmstadt, Germany) from monolayer cells initially grown on collagen type I–coated plastic to 60% confluence in standard medium containing 10% FBS and then maintained for an additional 48 hours in medium containing 1% FBS. In the case of transforming growth factor (TGF)-β, cell lysates were from cultures maintained for 48 hours in medium with 1.0% FBS. Blotting conditions were optimized for each protein biomarker, with primary/secondary antibody dilutions indicated as follows: cytokeratin-19 (CK19), 1:1000/1:3000; α-smooth muscle actin (α-SMA), 1:750/1:5000; desmin, 1:500/1:3000; thymocyte differentiation antigen-1 (Thy-1), 1:100/1:3000; elastin, 1:750/1:5000; nucleoside triphosphate diphosphohydrolase 2 (NTPDase2), 1:200/1:5000; mesothelin (Msln), 1:1000/1:3000; periostin (Postn), 1:1000/1:3000; TGF-β, 1:500/1:3000. Protein band intensity values normalized to actin or to vinculin are from measurements made on three separate Western blots per biomarker protein. B: Representative photomicrographs demonstrate strong immunoreactivity for Msln in the cholangiocarcinoma cells (arrows) and for Postn specifically in the stroma of desmoplastic cholangiocarcinoma (ICC) formed in rat liver orthotopically transplanted with TDECC cells. Corresponding pair-matched right liver lobe tissue exhibit a normal liver histology (NL) sampled from the same tumor bearing rats and do not immunostain for either Msln or Postn. Histological sections were counterstained with hematoxylin. Primary antibody dilutions shown in parentheses. Data are expressed as means ± SD (A and B). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Scale bars = 50 μm.

Article Snippet: 26 , 27 , 28 Immunofluorescence biomarker profile staining of the individual cholangiocarcinoma and fibroblastic cell strains cultured on collagen type I–coated coverslips and fixed for approximately 10 minutes with ice-cold 4% paraformaldehyde in phosphate-buffered saline, pH 7.4, was performed using the following primary antibodies: CK19 mouse monoclonal (VP-C415, b170) (Vector Laboratories, Inc., Burlingame, CA), α-SMA mouse monoclonal (M0851, 1A4) (Dako Cytomation, Inc., Carpinteria, CA), desmin polyclonal goat IgG (sc-7559, Y-20), vimentin (Vim) mouse monoclonal (sc-6260, V9), thymocyte differentiation antigen-1 (Thy-1) mouse monoclonal (sc-53116, OX7), CD34 mouse monoclonal (sc-7324, ICO115), and CD45 mouse monoclonal (sc-53047, OX30) (Santa Cruz Biotechnology, Inc., Paso Robles, CA), anti-rat elastin rabbit polyclonal IgG (CL55041AP, Lot 333K) (Cedarlane Labs, Burlington, NC), and anti-Postn rabbit polyclonal IgG (ab14041) (Abcam, Inc., Cambridge, MA).

Techniques: Western Blot, Cell Culture, Biomarker Assay

Journal: The American Journal of Pathology

Article Title: Transforming Growth Factors α and β Are Essential for Modeling Cholangiocarcinoma Desmoplasia and Progression in a Three-Dimensional Organotypic Culture Model

doi: 10.1016/j.ajpath.2017.01.013

Figure Lengend Snippet: A: 3D gel co-culturing of TDECC with TDFSM cells over a 4-day period significantly increases the accumulation of α-SMA–positive fibroblastic cells within the gel matrix over those determined over the same time period for TDFSM mono-cell cultures. B: 3D gel co-culturing of TDFSM cells with TDECC cells significantly increases the number of CK19-positive cholangiocarcinoma structures/cm2 section area detected in random histological sections from these gel cultures over those determined for TDECC cells cultured alone. The initial viable cell plating density/gel for TDECC cells was 2 × 105 and for TDFSM, 8 × 105. The FBS concentration in each culture was reduced from 10% to 1.0% at 24 hours after the initial cell plating and the cultures analyzed 72 hours later. Data are expressed as means ± SD (A and B). n = 3 cultures analyzed per data point, with measurements made on random fields of 6 sections/culture (A and B). ∗P < 0.05, ∗∗P < 0.01. Scale bars = 100 μm (A and B).

Article Snippet: 26 , 27 , 28 Immunofluorescence biomarker profile staining of the individual cholangiocarcinoma and fibroblastic cell strains cultured on collagen type I–coated coverslips and fixed for approximately 10 minutes with ice-cold 4% paraformaldehyde in phosphate-buffered saline, pH 7.4, was performed using the following primary antibodies: CK19 mouse monoclonal (VP-C415, b170) (Vector Laboratories, Inc., Burlingame, CA), α-SMA mouse monoclonal (M0851, 1A4) (Dako Cytomation, Inc., Carpinteria, CA), desmin polyclonal goat IgG (sc-7559, Y-20), vimentin (Vim) mouse monoclonal (sc-6260, V9), thymocyte differentiation antigen-1 (Thy-1) mouse monoclonal (sc-53116, OX7), CD34 mouse monoclonal (sc-7324, ICO115), and CD45 mouse monoclonal (sc-53047, OX30) (Santa Cruz Biotechnology, Inc., Paso Robles, CA), anti-rat elastin rabbit polyclonal IgG (CL55041AP, Lot 333K) (Cedarlane Labs, Burlington, NC), and anti-Postn rabbit polyclonal IgG (ab14041) (Abcam, Inc., Cambridge, MA).

Techniques: Cell Culture, Concentration Assay

Journal: The American Journal of Pathology

Article Title: Transforming Growth Factors α and β Are Essential for Modeling Cholangiocarcinoma Desmoplasia and Progression in a Three-Dimensional Organotypic Culture Model

doi: 10.1016/j.ajpath.2017.01.013

Figure Lengend Snippet: A: Treatment of TDECC + TDFSM 3D co-cultures with LY2157299 significantly inhibits the accumulation of α-SMA–positive fibroblastic cells/cm2 section area in a concentration-dependent manner. B: Likewise, the LY2157299 treatment also significantly reduces the number of CK19-positive cholangiocarcinoma cell spheroid/ductal structures/cm2 section area formed within the TDECC + TDFSM co-cultures. Arrows in A and B exemplify each cell type/structure within the gel matrix over those of the vehicle control (VC) cultures. Initial viable cell plating density for TDECC = 2 × 105/gel and for TDFSM = 8 × 105/gel. Beginning at 24 hours after initial cell plating, cultures containing 1% FBS were treated daily with LY2157299 in dimethyl sulfoxide over a 72-hour period and analyzed on day 4. Data are expressed as means ± SD. n = 3 cultures analyzed for each indicated concentration level, with α-SMA–positive cell counts made on three random histological sections per culture and those of CK19-positive cell structures made on 2 random sections per culture. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus control. Scale bars = 100 μm.

Article Snippet: 26 , 27 , 28 Immunofluorescence biomarker profile staining of the individual cholangiocarcinoma and fibroblastic cell strains cultured on collagen type I–coated coverslips and fixed for approximately 10 minutes with ice-cold 4% paraformaldehyde in phosphate-buffered saline, pH 7.4, was performed using the following primary antibodies: CK19 mouse monoclonal (VP-C415, b170) (Vector Laboratories, Inc., Burlingame, CA), α-SMA mouse monoclonal (M0851, 1A4) (Dako Cytomation, Inc., Carpinteria, CA), desmin polyclonal goat IgG (sc-7559, Y-20), vimentin (Vim) mouse monoclonal (sc-6260, V9), thymocyte differentiation antigen-1 (Thy-1) mouse monoclonal (sc-53116, OX7), CD34 mouse monoclonal (sc-7324, ICO115), and CD45 mouse monoclonal (sc-53047, OX30) (Santa Cruz Biotechnology, Inc., Paso Robles, CA), anti-rat elastin rabbit polyclonal IgG (CL55041AP, Lot 333K) (Cedarlane Labs, Burlington, NC), and anti-Postn rabbit polyclonal IgG (ab14041) (Abcam, Inc., Cambridge, MA).

Techniques: Concentration Assay

Journal: JAMA Facial Plastic Surgery

Article Title: Use of Condensed Nanofat Combined With Fat Grafts to Treat Atrophic Scars

doi: 10.1001/jamafacial.2017.1329

Figure Lengend Snippet: A and B, Under Fontana-Masson staining, an increase in melanin is seen in the basal cell layer between the preoperative (A) and 6-month postoperative (B) specimens (original magnification ×400). C and D, Under cytokeratin (CK) 14 staining, almost no sebaceous or sweat glands were observed preoperatively (C), but they were clearly visualized 6 months postoperatively (D) (original magnification ×200). E and F, Under CK19 staining, almost no sebaceous or sweat glands were observed preoperatively (E), but they were clearly visualized 6 months postoperatively (F) (original magnification ×100).

Article Snippet: Following citrate incubation and blocking with 5% rabbit serum for 30 minutes, slides were incubated at 4°C overnight with the following antibodies against cytokeratin (CK) 14 and CK19 (both at 1:200 concentration; ProteinTech).

Techniques: Staining

Journal: World journal of clinical cases

Article Title: Gradient inflammation in the pancreatic stump after pancreaticoduodenectomy: Two case reports and review of literature.

doi: 10.12998/wjcc.v12.i9.1649

Figure Lengend Snippet: Figure 1 Pathology in the pancreatic stump with postoperative pancreatic fistula after pancreaticoduodenectomy. A: Subserosal hematoma; B: Infiltration of inflammatory cells in glandular lobes and interlobular structures; C: Necrotic foci; D: Concentration of inflammatory cells and red blood cells in the main pancreatic duct; E: Destruction of inflammatory cells in the interlobular duct; F: Decomposition of inflammatory cells in the acinar duct metaplasia (ADM)-formed ducts; G: Transmigration of a neutrophil through the ADM-formed duct; H: Weakening or disappearance of apoptosis in the pancreatic stump after pancreatico- duodenectomy (transferase dUTP nick-end labeling); I: Normal level of apoptosis in control tissue of the pancreatic head before the postoperative pancreatic fistula (transferase dUTP nick-end labeling); J: Normal level of cytokeratin 19 (CK19) expression in the pancreas; K: Strong expression of CK19 in the pancreatic stump after pancreaticoduodenectomy; L: Malformation of the ADM-formed duct; M: Dilatation of the ADM-formed ducts.

Article Snippet: The presence of pancreatic ducts and centroacinar cells was detected using a mouse anticytokeratin 19 (CK19) antibody (ZM-0074; OriGene Technologies), followed by a secondary antibody using the ultraView Universal Diaminobenzidine (DAB) Detection Kit (Roche).

Techniques: Concentration Assay, Transmigration Assay, End Labeling, Control, Expressing

Journal: EMBO Molecular Medicine

Article Title: Comprehensive molecular characterization of collecting duct carcinoma for therapeutic vulnerability

doi: 10.1038/s44321-024-00102-5

Figure Lengend Snippet: ( A ) Workflow for establishing PDX and primary cell line; and screening for potential drug targets. ( B ) Hematoxylin and eosin (H&E), CK19, and PAX8 staining of CDC1 patient and PDX samples. ( C ) Single-sample GSEA (ssGSEA) score for all RNA-seq samples. The top 15 cell types (ranked by the variance of ssGSEA scores) are shown. CDC1 (PDX) clusters together with tumors, different from normal samples. ( D ) Overall ranking of the 130 small molecule compounds in the drug library by their inhibition rates. ( E ) Top ten drug candidates with the highest inhibition rates. ( F ) Testing of the top ten drug candidates at 1 µM concentration in clear cell RCC cell lines (A-498 and 786-O) and normal immortalized kidney cell line (HK-2). ( G ) Dosage-dependent response of CDC1 to CDK9 inhibitor LDC000067 and its IC50s for different cell lines. The cells were treated with LDC000067 for 96 h. ( H ) Relative growth rate of CDC1 when treated with LDC000067 at 1 µM concentration. Data were presented as the mean ± SD ( n = 3). P value = 3.30E-05, two-way ANOVA. .

Article Snippet: The sections were then incubated with antibodies specific to Ki67 (Zsbio Commerce Store, cat #ZA-0502), p-AURKA (Cell Signaling Technology, cat #3079), TPX2 (Cell Signaling Technology, cat #12245), p-RB1 (Cell Signaling Technology, cat #8180), CK19 (Cell Signaling Technology, cat #13902) and PAX8 (Cell Signaling Technology, cat #28556) at 4 °C overnight, respectively.

Techniques: Staining, RNA Sequencing, Drug discovery, Inhibition, Concentration Assay

Journal: EMBO Molecular Medicine

Article Title: Comprehensive molecular characterization of collecting duct carcinoma for therapeutic vulnerability

doi: 10.1038/s44321-024-00102-5

Figure Lengend Snippet: Reagents and tools table

Article Snippet: The sections were then incubated with antibodies specific to Ki67 (Zsbio Commerce Store, cat #ZA-0502), p-AURKA (Cell Signaling Technology, cat #3079), TPX2 (Cell Signaling Technology, cat #12245), p-RB1 (Cell Signaling Technology, cat #8180), CK19 (Cell Signaling Technology, cat #13902) and PAX8 (Cell Signaling Technology, cat #28556) at 4 °C overnight, respectively.

Techniques: Sequencing, Drug discovery, Modification, Saline, cDNA Synthesis, Polymer, Software

Journal: Japanese journal of clinical oncology

Article Title: An optimal mRNA marker for OSNA (One-step nucleic acid amplification) based lymph node metastasis detection in colorectal cancer patients.

doi: 10.1093/jjco/hys227

Figure Lengend Snippet: Figure 1. (A) Study protocol. Half a lymph node was used for the study. Slices (i, ii and iii) were used for the histopathologic staining with H&E and/or AE1/AE3-IHC. Blocks (a and c) were used for QRT-PCR or OSNA. (B) A schematic diagram of the OSNA assay. (C) Time courses of RT-LAMP reaction using CK19 mRNA solutions of known concentration: open squares: 2.5 107 copy/mL; 4: 2.5 106 copy/ml; closed diamonds: 2.5 105 copy/ml; closed circles: 2.5 104 copy/ml; closed squares: 2.5 103 copy/ml; closed triangles: 2.5 102 copy/ml; open circles: negative control.

Article Snippet: The section was incubated at 48C in mouse monoclonal anti-human CK19 antibody (RCK108, Dako, Glostrup, Denmark), and stained with streptavidin–biotin immunoperoxidase (iVIEW DAB Detection Kit, Ventana, Arizona) according to the manufacturer’s instructions.

Techniques: Staining, Quantitative RT-PCR, Concentration Assay, Negative Control

Journal: Japanese journal of clinical oncology

Article Title: An optimal mRNA marker for OSNA (One-step nucleic acid amplification) based lymph node metastasis detection in colorectal cancer patients.

doi: 10.1093/jjco/hys227

Figure Lengend Snippet: Figure 2. Expression of mRNA markers in metastasis-positive and -negative LNs. Nine mRNA markers (CEA, CK19, FXYD3, CK20, MUC13, CFTR, MUC11, MUC12 and MUC2) were evaluated by QRT-PCR using 10 positive (closed circle) and 10 negative (open circle) LNs from 20 patients. A DCt values for each mRNA marker was obtained from the following equation: DCt ¼ Ct (for each mRNA) – Ct (for b-actin).

Article Snippet: The section was incubated at 48C in mouse monoclonal anti-human CK19 antibody (RCK108, Dako, Glostrup, Denmark), and stained with streptavidin–biotin immunoperoxidase (iVIEW DAB Detection Kit, Ventana, Arizona) according to the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Marker

Journal: Japanese journal of clinical oncology

Article Title: An optimal mRNA marker for OSNA (One-step nucleic acid amplification) based lymph node metastasis detection in colorectal cancer patients.

doi: 10.1093/jjco/hys227

Figure Lengend Snippet: Figure 3. Expression of CK19, CEA and CK20 mRNAs as measured by the OSNA assay. The arrows indicate the results when using the same LN lysate. The amplification was carried out for 16 min. When the amplification did not proceed, the result was categorized as ‘not detected’ (ND).

Article Snippet: The section was incubated at 48C in mouse monoclonal anti-human CK19 antibody (RCK108, Dako, Glostrup, Denmark), and stained with streptavidin–biotin immunoperoxidase (iVIEW DAB Detection Kit, Ventana, Arizona) according to the manufacturer’s instructions.

Techniques: Expressing

Journal: Japanese journal of clinical oncology

Article Title: An optimal mRNA marker for OSNA (One-step nucleic acid amplification) based lymph node metastasis detection in colorectal cancer patients.

doi: 10.1093/jjco/hys227

Figure Lengend Snippet: Figure 4. ROC curves for the OSNA assay with CK19, CEA and CK20 mRNAs. The sensitivities and specificities for the OSNA assay with CK19, CEA and CK20 mRNAs at respective cut-off values of 25, 50, 75, 100, 250, 500, 750, 1000, 2500, 5000 and 7500 copies/ml were calculated based on the data in Fig. 3.

Article Snippet: The section was incubated at 48C in mouse monoclonal anti-human CK19 antibody (RCK108, Dako, Glostrup, Denmark), and stained with streptavidin–biotin immunoperoxidase (iVIEW DAB Detection Kit, Ventana, Arizona) according to the manufacturer’s instructions.

Techniques: