Tissue was acquired from the Oxford Radcliffe Biobank with informed consent. The study was approved by the South Central-Oxford C Research Ethics Committee. Organoids were derived from human liver metastases of colorectal cancer using rapid isolation as previously described (Ashley et al., 2014 (link); Buzzelli et al., 2018 (link)). Organoids were grown in DMEM/F12 + GlutaMAX containing StemPro, ROCK inhibitor, R-Spondin-1 (RSPO-1), Noggin, WNT3A, EGF, Insulin-like Growth Factor 1 (IGF-1), Fibroblast Growth Factor 10 (FGF-10), Fibroblast Growth Factor basic (FGF-β), and Endothelin 3 (ET3). Expression of colonic markers were assessed in organoid cultures and original tumor specimens. All organoids showed similar expression of colonic markers as their respective tumor specimen confirming their origin (Buzzelli et al., 2018 (link)). Organoids were passaged 2 d before itraconazole treatment. For itraconazole treatment, organoids were given fresh media containing 1.25, 2.5, or 5 µM itraconazole or untreated media. Time-lapse images were captured every 3 h for 60 h on a Nikon Eclipse Ti-E inverted microscope system. Images were converted to TIF files and the area of organoids were measured using in-house software written in MATLAB R2015b software.
Eclipse ti e inverted microscope system
Manufactured by Nikon
Sourced in Japan
The Eclipse Ti-E is an inverted microscope system designed for advanced research applications. It features a stable and precise optical system, allowing for high-resolution imaging and analysis of samples. The Eclipse Ti-E is equipped with a wide range of accessories and configurations to support various imaging techniques, including brightfield, phase contrast, and fluorescence microscopy.
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Lab products found in correlation
Anti rabbit cy3, by Jackson ImmunoResearch (2 mentions)
Donkey anti rabbit alexa 488, by Thermo Fisher Scientific (1 mentions)
Af1062, by R&D Systems (1 mentions)
Alexa 594 donkey anti goat, by Thermo Fisher Scientific (1 mentions)
Mount fluorcare dapi, by Carl Roth (1 mentions)
Anti gfp ab6556, by Abcam (1 mentions)
Prolong mounting medium with dapi, by Thermo Fisher Scientific (1 mentions)
Fibronectin coated coverslips, by Merck Group (1 mentions)
Anti phospho hsp27 ser82, by Cell Signaling Technology (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Mowiol mounting medium, by Thermo Fisher Scientific (1 mentions)
Anti α actinin, by Merck Group (1 mentions)
Connexin 43, by Merck Group (1 mentions)
Zetasizer nano, by Malvern Panalytical (1 mentions)
Luna c18, by Phenomenex (1 mentions)
Tecnai t12 cryo electron microscope, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000, by Phenomenex (1 mentions)
9 protocols using eclipse ti e inverted microscope system
1
Colorectal Cancer Organoid Response to Itraconazole
2
Immunostaining of Cardiac Fibroblasts
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Sections prepared as described above were thawed, dried at room temperature (RT, 15 min) and fixed with ice-cold 2% paraformaldehyde (w/v, Carl Roth, Karlsruhe, Germany) in PBS (phosphate-buffered saline: 137 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 1.8 mM KH2PO4) for 6 min. After washing (3x PBS, 5 min, RT), slices were blocked with 5% (w/v) BSA (bovine serum albumin), 0.75 % Triton X-100 (v/v) in PBS (2 h, RT). Subsequently, slices were incubated with respective antibodies (anti-GFP, ab6556, 1:1000, abcam; anti-PDGF-receptor α, AF1062, 5 µg/ml, R&D-Systems; in PBS, 5 % BSA, 0.025 % Triton X-100; overnight at 4 °C) to detect cGi-500 and identify cardiac fibroblasts42 (link), respectively. After washing (3x PBS, 5 min, RT), slices were incubated with secondary antibodies (Alexa488 donkey anti-rabbit, A21206, Thermo Fisher, 1:1000; Alexa594 donkey anti-goat, A11058, Thermo Fisher, 1:1000; in PBS, 5% BSA, 0.025% Triton X-100; 2 h, RT, dark). After washing (3x PBS, 5 min, RT), the slices were covered in aqueous embedding solution (Mount FluorCare DAPI, Carl Roth, Karlsruhe, Germany) and analysed by confocal laser scanning microscopy (Nikon Eclipse Ti-E Inverted Microscope System). Laser and microscope settings were kept identical for different genotypes.
3
Cellular Uptake of miR-196a I Nanocomposites
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Cy3-labeled miR-196a I (Cy3-miR-196a I, red) was used for uptake and DAPI (blue) for re-staining. HepG2 cells were treated with miR-196a I, miR-196a I/DEAC-PNAG, and miR-196a I/DEAC-PNAG-FA nanocomposites with a molar concentration of 60 nM for 4 h. The cells were washed with PBS and fixed with 4% paraformaldehyde (PFA) for 20 min. After the PFA removal, DAPI FluoroQuest™ with an anti-fading fixing medium (AAT Bioquest, Inc., USA) was used and placed on a glass slide. Fluorescence images were obtained using an Eclipse Ti-E Inverted Microscope System (Nikon, Japan).
4
Immunofluorescence Staining of Adherent Cells
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Cells were seeded in fibronectin-coated coverslips (Sigma Aldrich). When desired, cells were fixed in 2% paraformaldehyde for 15 min and permeabilized with 0.5% Triton X-100 for 10 min at room temperature. Blocking step was made in 3% Bovine Serum Albumin (BSA) for 1 h. Cells were incubated overnight at 4 °C with the primary antibody diluted in blocking buffer. Next day, secondary antibodies were incubated for 1 h at room temperature in the dark. Antibodies and dilutions are listed in Supplementary Table 7 . Finally, cells were mounted in Prolong Mounting Medium with DAPI (Invitrogen) and images were taken in a Nikon Eclipse Ti-E inverted microscope system. For quantification, at least 200 cells per staining were evaluated using ImageJ software.
5
Cell Detachment and Wound Healing Assays
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For cell detachment assay, cells were grown in 96-well plate for 24 h and then treated with 1 μg/ml tetracycline to induce p63 expression for 24 or 48 h. After washing in 0.5 % EGTA in PBS, cells were incubated in trypsin solution (0.025 % trypsin and 0.5 % EGTA in PBS) for the indicated periods of time. Trypsinization was stopped by adding 100 μl DMEM with 10 % FBS. Cells were then washed in PBS, fixed in 4 % paraformaldehyde for 10 min, washed again in PBS and stained with crystal violet (5 mg/ml). After washing in tap water, cells were dried for 30 min at RT and then 2 % SDS was used to dissolve the colour. Absorbance was measured at 595 nm. Three independent experiments were performed, each sample was in hexaplicate.
For wound healing assay, cells were seeded in 6-well plates and cultured until confluent. Scratches were made using pipette tips and phase contrast images were taken with Eclipse Ti-E inverted microscope system (Nikon, Japan) for 48 h at 1 h intervals.
For wound healing assay, cells were seeded in 6-well plates and cultured until confluent. Scratches were made using pipette tips and phase contrast images were taken with Eclipse Ti-E inverted microscope system (Nikon, Japan) for 48 h at 1 h intervals.
6
Immunostaining Frozen Cardiac Tissue
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The frozen LV tissue slides were air-dried for 10 min, rehydrated with PBS and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 5 min. Then the slides were washed three times for 5 min with PBS. Afterwards the tissue was incubated with wheat germ agglutinin (WGA) for 5 min and washed three times for 5 min with PBS. Next, the slides were blocked with 5% BSA/PBS and 0.1% Triton for 1 h at room temperature. After washing 3× times with PBS, the primary antibody was diluted in 5% BSA/PBS and incubated overnight at 4 °C. Subsequently, the slides were incubated with the secondary antibody diluted in 5% BSA/PBS overnight at 4 °C. The slides were then washed again three times with PBS, covered with ultrathin glass coverslips and sealed with Mowiol mounting medium and ultrathin glass coverslips overnight at 4 °C. For the imaging, confocal laser scanning microscopy (cLSM) (Nikon Eclipse Ti-E inverted Microscope System, Nikon Instruments, Nicon Corp, Shinagawa, Tokyo, Japan) was used.
7
Dual-Immunostaining of Cardiac Tissue
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Frozen histological LV sections (n = 3/group) were fixed, blocked and dual-stained with anti-PKD (Sigma-Aldrich; dilution 1:200) or anti-phospho-HSP27 (Ser 82) (Cell Signaling Technology; 1:50) and anti-α-actinin (sarcomere; Sigma-Aldrich; dilution 1:400) antibody, and were incubated with the appropriate secondary antibodies: (FITC) anti-mouse (Rockland Immunochemicals Inc, Limerick, PA, USA; dilution 1:300) and Cy3 anti-rabbit (Jackson ImmunoResearch Laboratories Inc, West Grove, PA, USA; dilution 1:100). Immunostained samples were analyzed by confocal laser scanning microscopy (Nikon Eclipse Ti-E Inverted Microscope System; Nikon Instruments, Nikon Corp, Shinagawa, Tokyo, Japan). Immunofluorescence imaging was processed equally among groups, for 2D intensity histogram analysis the Coloc2 plugin in FIJI was used.
8
Cardiac Protein Localization and Quantification
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Frozen LV unfixed slides (n = 3 samples/heart) were air-dried for 10 min and fixed in 4% paraformaldehyde in phosphate buffered saline (PBS; Sigma-Aldrich). After washing 3 times in PBS for 5 min, tissue was blocked in 5% bovine serum albumin (Sigma-Aldrich) in PBS for 1 h at room temperature. After further washing 3 times in PBS for 5 min, fixed slides were dual-stained with anti-guanylyl cyclase β1 ER-19 (Sigma-Aldrich; dilution 1:200) or Connexin 43 (Sigma-Aldrich; dilution 1:400/Thermofisher Scientific; dilution 1:200) antibodies and anti-α-actinin (sarcomere; Sigma-Aldrich; dilution 1:400) overnight at 4°C. After washing in PBS, slides were subsequently incubated overnight with secondary antibodies: fluorescein (FITC) anti-mouse (Rockland Immunochemicals Inc., Limerick, PA, USA; dilution 1:300) and Cy3 anti-rabbit (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA; dilution 1:100). After multiple washings slides were covered and sealed by Mowiol mounting medium and ultrathin glass coverslips (Thermo Fisher Scientific). Immuno-stained samples were analyzed by confocal laser scanning microscopy (cLSM) (Nikon Eclipse Ti-E Inverted Microscope System; Nikon Instruments, Nikon Corp, Shinagawa, Tokyo, Japan). Immunofluorescence imaging parameters were identical among groups.
9
Comprehensive Nanoparticle Characterization
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