Sections were prepared from the abovementioned resected specimens (4 μm). Immunohistochemical (IHC) staining for LRG was performed using a rabbit anti‐LRG monoclonal antibody (1:250, ab178698">ab178698 ; Abcam, Chicago, IL, USA), a rabbit anti‐Smad4 monoclonal antibody (1:200, ab40759; Abcam), a rabbit anti‐Smad2 polyclonal antibody (1:100, ab53100; Abcam), a mouse anti‐E‐cadherin polyclonal antibody (610181, 1:200; GE Healthcare Biosciences, Piscataway, NJ, USA) and a mouse anti‐vimentin monoclonal antibody (V6630, 1:200; Sigma‐Aldrich, St. Louis, MO, USA) overnight at 4°C, with visualization using Envision ChemMate (Dako, Glostrup, Denmark), according to the manufacturer's protocol. Three independent gastroenterological oncologists (HW, SK and TO), who were blinded to the histologic data, analyzed the stained sections, which were also photographed using a light microscope (DM2500 with the Leica Application Sweat software program [version 3.80]; Leica Microsystems GmbH, Wetzlar, Germany).
V6630
Manufactured by Merck Group
Sourced in United States
The V6630 is a laboratory centrifuge designed for general-purpose applications. It features a compact and robust construction, enabling efficient separation of samples. The centrifuge can accommodate a variety of rotor types and sample volumes, making it a versatile instrument for various research and diagnostic settings.
Automatically generated - may contain errors
Lab products found in correlation
Mab353, by Merck Group (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Ab178698, by Abcam (1 mentions)
Ab40759, by Abcam (1 mentions)
Chemmate envision, by Agilent Technologies (1 mentions)
Ab53100, by Abcam (1 mentions)
Ab16051, by Abcam (1 mentions)
Ab108630, by Abcam (1 mentions)
Ab134047, by Abcam (1 mentions)
N cadherin sc 7939, by Santa Cruz Biotechnology (1 mentions)
Sc 8312, by Santa Cruz Biotechnology (1 mentions)
Eif2α sc 133132, by Santa Cruz Biotechnology (1 mentions)
T5168, by Merck Group (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Mab360, by Merck Group (1 mentions)
200 inverted fluorescent microscope, by Zeiss (1 mentions)
Leica tcs sp5 scanning laser confocal fluorescence microscope, by Leica Microsystems (1 mentions)
M1406, by Merck Group (1 mentions)
17 protocols using v6630
1
Immunohistochemical Analysis of Gastric Cancer
2
Protein Expression Analysis Protocol
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TNF-α was purchased from PeproTech (Rocky Hill, NJ, USA). Primary antibodies specific for α-tubulin (T5168) and vimentin (V6630) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies specific for VCAM-1 (ab134047), MGMT (ab108630), and β-catenin (ab16051) were purchased from Abcam (Cambridge, UK). Primary antibodies specific for AKT1/2/3 (sc-8312), p-AKT1/2/3 (Ser473, sc-7985-R), connexins 43 (sc-271837), eIF2α (sc-133132), and N-cadherin (sc-7939) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibodies specific for EGFR (4267), p-Cx43 (Ser368, 3511), and p-mTOR (Ser2448, 2971) were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary antibodies specific for p-GSK3β (Tyr216, 44604G) was purchased from Invitrogen (Carlsbad, CA, USA).
3
Characterizing Neural Progenitor Cell Markers
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The self-renewal and differentiation markers of the NPCs were also assessed by immunofluorescence34 (link). For immunofluorescence staining analysis cells were incubated with the primary antibody Nestin (1:400; MAB353, Millipore), Tuj1 (1:500,05-549, Upstate), Sox2 (1:100; 481400, Life technologies), Map2(1:400; M1406, Sigma), Vimentin (1:100; V6630, Sigma), GFAP (1:500; MAB360, millipore) overnight at 4 °C. The secondary antibodies are anti-mouse IgG FITC antibody (1:200, St. Louis, MO, USA) and anti-rabbit IgG FITC antibody (1:1000, St. Louis, MO, USA) diluted in blocking buffer. Nuclei are counter-stained with Hochest 33342 (1:500; 94403, St. Louis, MO, USA). The fluorescent images of 2-D cultured cells were visualized on a Zeiss 200 inverted fluorescent microscope (Carl Zeiss, Jena, Germany). The number of immunostained cells was counted in each of three random fields per well and the fluorescence images were selected randomly. The quantification of the immunofluorescence signal was performed by Image-Pro Plus software (Media Cybernetics, Bethesda, MD). The fluorescent images of 3-D cultured cells were taken with a Leica TCS SP5 scanning laser confocal fluorescence microscope (Leica Microsystems, Inc., Germany).
4
Immunofluorescence Analysis of Caspase-1 and NF-κB in Fixed EC
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EC were seeded on microscope glass and fixed for 20 min in a 4% (v/v) paraformaldehyde solution. Cell permeabilization was performed with 0.1% (v/v) Triton X-100 in PBS for 10 min. Caspase-1 and NF-κB immunofluorescence was performed by overnight incubation with specific antibodies (1:500) followed by 1 h incubation with Alexa Fluor 633 (1:1000, A21071, Invitrogen, Waltham, MA, USA). For cytoskeleton staining anti-vimentin antibody (1:1000, V6630, Sigma Aldrich, St. Louis, MO, USA) and Alexa Fluor 488 (1:1000, A32723, Invitrogen, Waltham, MA, USA) secondary antibody were used, while nuclear staining was performed for 7 min with 2.5 µg/mL 4′,6-diamidino-2-phenylindole (DAPI; Sigma Aldrich, St. Louis, MO, USA). Immunofluorescence analysis was performed with a confocal microscope (model LSM 700, Zeiss, Oberkochen, Germany) equipped with a plan apochromat X63 (NA1.4) oil immersion objective, and fluorescence intensity, reported as arbitrary fluorescence units (AFU), was estimated with ImageJ 1.52 n software (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA).
5
Immunocytochemical analysis of differentiated cells
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Differentiated cells from embryoid bodies were fixed on dishes with 4% PFA overnight at 4°C, then washed 3 times with PBS(-) for 5 min. Expression of α-SMA, β-tubulin or AFP in cultured cells was detected with the indicated antibody: anti-α-SMA antibody (1:200; V6630, Sigma, Tokyo, Japan); anti-β-tubulin antibody (1:100 T4026; SIGMA); anti-AFP antibody (1:100 MAB1368; R&D Systems, MN, USA). Binding was visualized with a secondary antibody labeled with Alexa Fluor 488 (1:500; A11008, Life Technologies) or Alexa Fluor 594 (1:500; A11062, Life Technologies), and cells were counterstained with DAPI (D1306, Life Technologies). An Olympus IX71 fluorescence microscope was used for fluorescent observation.
6
Protein Expression Analysis in Cells
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A Pierce BCA protein assay kit (Thermo Fisher Scientific) was used to determine the protein concentration of lysed samples against Bovine serum albumin standards of known concentration. Samples containing equal amounts of protein were electrophoresed on SDS–PAGE gels, and transferred to nitrocellulose membrane. IF1 (ab110277, Abcam), ATPsβ (ab14730, Abcam), Calnexin (C4731, Sigma) and Vimentin (V6630, Sigma) primary antibodies, and peroxidase-conjugated anti-mouse (A5278, Sigma) and anti-rabbit (A6154, Sigma) secondary antibodies were used for the immunoblotting assays. Bands were visualized with enhanced chemiluminescence (ECL) Western blotting substrates (ThermoScientific) and a ChemiDoc XRS + molecular imaging system (Bio-Rad). The pixel intensities of the bands were calculated using ImageLab software.
7
Protein Expression Analysis of HBEC3 and T2-HBEC3
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Whole cell extracts of HBEC3 and T2-HBEC3 were prepared and protein concentrations were measured using the BCA Assay (Thermo Fisher). Protein samples (25 µg) were run on 10% Mini-Protean TGX Stain-Free gel (BioRad, Oslo, Norway) and transferred to a PVDF membrane (BioRad). Antibodies against vimentin (V6630, Sigma-Aldrich), β-actin (MA5-11869, Thermo Fisher Scientific) and E-cadherin (EP700Y, Abcam, Cambride, United Kingdom) were used. Secondary antibodies were horseradish peroxidase-conjugated antirabbit/antimouse IgG antibodies (Cell Signaling Technology, Leiden, The Netherlands). Immunoreactive bands were detected using chemiluminescent substrate (SuperSignal West Pico, Thermo Fisher Scientific).
8
Immunocytochemistry of ARPE-19 Cells
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ARPE-19 e Y-negative clone 3 cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 and saturated in 3% BSA. Cells were incubated with mouse anti-vimentin antibody (Sigma-Aldrich Cat# V6630, RRID:AB_477627; diluted 1:40 in 1% BSA), followed by secondary fluorescent Alexa Fluor 488 conjugated antibody incubation (Thermo Fisher Scientific Cat# A-11001, RRID:AB_2534069; diluted 1:400 in 1% BSA). The coverslips were mounted on glass microscope slides with ProLong Gold with DAPI reagent (Thermo Fisher Scientific), to counterstain nuclei. Images were acquired using Olympus IX71 microscope with CoolSNAP ES camera. Experiments were repeated at least two times.
9
Quantitative Protein Expression Analysis
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Western blot analysis was performed using antibodies against vimentin (1:1000, V6630, Sigma‐Aldrich), E‐cadherin (1:1000, Cell Signalling Technology, Danvers, MA), EGFR (1:100, Cell Signalling Technologies) and β‐actin (1:5000, Sigma‐Aldrich). The signals obtained were visualised with the enhanced chemiluminescence (ECL) detection system (Amersham) and normalised to β‐actin signal.
10
Immunohistochemical Analyses of Vimentin and Cytokeratin
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Immunohistochemical analyses were performed on 10-μm-thick frozen tissue sections using indirect immunofluorescence. Primary antibodies to vimentin (1:1000; V6630, Sigma-Aldrich) and pan-cytokeratin (1:1000; F3418, Sigma-Aldrich) were used in the analyses. Goat anti-mouse IgG conjugated with Alexa 488 (1:1000; A11029, Thermo Fisher Scientific) and goat anti-mouse IgG conjugated with Alexa 568 (1:400; A11031, Thermo Fisher Scientific) were used to detect primary antibodies. Fluoromount-G™, with 4′6-diamidino-2-phenylindole (DAPI; 00-4959-52, Thermo Fisher Scientific), was used to visualize nuclei and as mounting medium. Processed tissue sections were examined and images were captured with a Nikon Eclipse 80i upright microscope equipped with a charge-coupled device camera (Nikon DS-Fi3).
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