Fresh pancreatic or subcutaneous tumors were rinsed in PBS and placed in Zinc Formalin Fixative (Sigma-Aldrich) overnight. Then they were dehydrated serially in 70%, 95%, and 100% ethanol. The tissues were then embedded in paraffin, which were processed to generate H&E and unstained sections. To rehydrate for immunofluorescence, unstained sections were serially submerged in Xylene (Sigma), 100% ethanol, 95% ethanol, 70% ethanol, and PBS. Sections were then blocked with 10% donkey serum + 0.1% TritonX-100 in PBS overnight at 4°C; followed by incubation with a goat anti-GFP antibody (Abcam, ab6673; 1:200) and a rat anti-mouse Ly6G antibody (BioXCell, BE0075-1; 1:200) for 1 hour at room temperature. After washing, the sections were incubated with Alexa Fluor 488 donkey anti-goat IgG (Invitrogen, A-11055; 1:200), Alexa Fluor 488 or Alexa Fluor 594 donkey anti-rat IgG (Invitrogen, A-21208 or A-21209; 1:200), and DAPI (Biolegend; 1:1000) for 1 hour at RT in the dark. Sections were then washed and mounted with Aqua-Poly/Mount (Polysciences). A Nikon Eclipse Ti-U fluorescent microscope was used to acquire images at a 64-bit data depth.
Eclipse ti u fluorescent microscope
Manufactured by Nikon
Sourced in Japan
The Eclipse Ti-U is a fluorescent microscope designed for advanced imaging applications. It features a modular design that allows for the integration of various accessories and components to suit different research needs. The core function of the Eclipse Ti-U is to provide high-quality, high-resolution fluorescent imaging capabilities for a wide range of samples and applications.
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Lab products found in correlation
Spot rt3, by Nikon (2 mentions)
Spot rt3 camera, by Nikon (2 mentions)
Digital sight ds u3 camera, by Nikon (2 mentions)
Ti u camera, by Nikon (2 mentions)
Zinc formalin fixative, by Merck Group (1 mentions)
Alexa fluor 488 donkey anti goat igg, by Thermo Fisher Scientific (1 mentions)
Ab6673, by Abcam (1 mentions)
Be0075 1, by BioXCell (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by BioLegend (1 mentions)
Xylene, by Merck Group (1 mentions)
Aqua poly mount, by Polysciences (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Cls3422 48ea, by Corning (1 mentions)
Ds qi2 digital camera, by Nikon (1 mentions)
Cellstain double staining kit, by Dojindo Laboratories (1 mentions)
Dihydroethidium assay kit, by Beyotime (1 mentions)
Mitotracker, by Cell Signaling Technology (1 mentions)
Nis elements software, by Nikon (1 mentions)
Stemi 508 stereo, by Zeiss (1 mentions)
2 7 dichlorofluorescein, by Merck Group (1 mentions)
21 protocols using eclipse ti u fluorescent microscope
1
Immunofluorescence Analysis of Tumor Sections
2
Transwell-Based Cell Migration Assay
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Cell migration was assayed using 24-well TranswellTM chambers with 6.5 mm-diameter polycarbonate filters with 8 μm-pore-size TranswellTM migration inserts according to the manufacturer’s instructions with the following modifications (Corning CLS3422-48EA, Glendale, AZ, USA). The assay was based on chemotactic directional migration. Cells were treated under experimental conditions for 48 h, as were the control cells. Then, the cells were trypsinized, washed twice with serum-free medium and counted. The same numbers of living cells were seeded (2.5−3 × 104 cells/well depending on the cell line) on the upper chamber and allowed to migrate through the filter to the lower chamber containing DMEM with 10% FBS. After 8–18 h (for U251MG and U87MG cells, respectively), the cells were fixed with 4% PFA for 15 min and stained with 0.5% crystal violet (Sigma-Aldrich) for 7–10 min. Cells that did not migrate through the filter were removed from the upper chamber by using a cotton swab. Migrated cells were photographed using a Nikon Eclipse Ti-U fluorescent microscope equipped with a 20× objective and DS-Qi2 digital camera and then counted with the Fiji distribution of the ImageJ 1.52a software (National Institutes of Health and the University of Wisconsin). The percentage of migrated cells was calculated by assuming 100% for the control conditions.
3
Measuring Mitochondrial Membrane Potential
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The mitochondrial membrane potential (ΔΨm) was measured using the mitochondrial membrane potential—specific fluorescent probe, JC-1 (Molecular Probes). H9c2 cells were seeded on glass coverslips and cultured at least overnight before the experiment. After treatment, cells were washed twice in ice-cold PBS and then incubated for 5 min at 37°C in modified Krebs-Henseleit solution (118mM NaCl, 5mM KCl, 1.2 mM KH2PO4, 25 mM NaHCO3, 5mM glucose, 1.2 mM MgSO4x7H2O) containing 0.1 μM JC-1. When excited at 488 nm, the dye emits green fluorescence (530 nm) at low ΔΨm and red (590 nm) at high ΔΨm. Following incubation, the cells were washed once with Krebs-Henseleit solution and then imaged with a Nikon Eclipse Ti-U fluorescent microscope equipped with a Spot RT3 camera using a 60x objective and epifluorescent illumination. The investigator collecting data was blinded to treatment groups. All experiments were repeated in triplicate.
4
Crataegus Extract Effects on Cell Morphology
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Cells were seeded at a density of 3 × 105 cells per 6-cm culture dish (Sarstedt, Nümbrecht, Germany). After 18 h, cells were treated with Crataegus extracts (250 µg/mL) for 48 h. Non-treated cells were used as a control. Cells incubated with DMSO at concentrations of 0.02–1.5% dependent on the flower extract concentrations (see Figure S1 ) served as an additional control for the flower extract analysis. Cell morphology was analyzed by light microscopy (Nikon Eclipse Ti-U fluorescent microscope equipped with a 20× objective and DS-Qi2 digital camera, Tokyo, Japan).
5
NF-κB Translocation Assay in HaCaT Cells
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HaCaT keratinocytes were plated onto coverslips and treated with daphnetin for 24 h, then stimulated with M5 cytokines for 30 min. Cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 10 min. After being washed with PBS for three times, the cells were permeabilized with PBS containing 0.5% Triton X-100 for 15 min, and subsequently were blocked with PBS containing 1% bovine serum albumin (BSA) at room temperature for 30 min. Next, the coverslips were incubated with rabbit anti-p65 (1:500) primary antibody overnight at 4 °C. After being washed with PBS for three times, the coverslips were incubated with Alexa Fluor 555-labeled anti-rabbit secondary antibody (1:1000) for 2 h. Hoechst dye 33,258 was used to stain nucleus for 4 min. After that, the coverslips were washed with PBS for three times and mounted onto the microscope slides. Immunofluorescence was observed using Nikon Eclipse Ti-U fluorescent microscope.
6
Islet Viability Evaluation via Cellstain Staining
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The Cellstain Double Staining Kit (Dojindo, Kumamoto, Japan), consisting of calcein acetoxymethyl and propidium iodide, was used to analyze islet viability according to the manufacturer's instructions. Samples were observed on an Eclipse Ti–U fluorescent microscope (Nikon, Tokyo, Japan). Islet viability was determined by counting viable (yellow-green) and non-viable (red) cells. The degree of cell viability was assessed as previously described [25 (link)]. Total viability was calculated by dividing the number of viable cells by the total number of cells assessed.
7
Quantifying Cellular ROS using DHE
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Dihydroethidium assay kit (Beyotime, Shanghai, China) was used to detect the level of ROS in Hela cells. The working solution Dihydroethidium (5 μM) was added to the Hela cells for 30 min after the treatments. Imaging was performed with a Nikon Eclipse Ti-U fluorescent microscope.
8
Quantifying Mitochondrial Membrane Potential
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ΔΨ was measured using the ΔΨ specific fluorescent probe, JC-1. WRL-68 or U-251 MG cells were seeded to glass coverslips and cultured at least overnight before the experiment. After the indicated treatment, cells were washed twice in ice-cold PBS, then incubated for 15 minutes at 37°C in modified Krebs-Henseleit solution containing 100 ng/mL of JC-1. When excited at 490 nm, the dye emits either green fluorescence at a low Δψ or red fluorescence at a high Δψ. Following incubation, the cells were washed once with modified Krebs-Henseleit solution, then visualized by a Nikon Eclipse Ti-U fluorescent microscope which was equipped with a Spot RT3 camera, using a 40x objective lens with epifluorescent illumination. All experiments were repeated three times.
9
Quantifying Cell Fusion Dynamics
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After cell differentiation, cells were stained with 50 nM MitoTracker (Cell Signaling Technology) for 45 min at 37 °C. Cells were then fixed with cold methanol for 10 min, followed by washing 3× with PBS for 5 min. Cells were permeabilized with 0.05% Triton X-100 solution, blocked with 1% bovine serum albumin solution for 30 min, and incubated overnight at 4 °C with the primary antibody. Cells were then washed 3× with PBS for 5 min and incubated with a goat anti-mouse immunoglobulin G, Flamma® 488-labeled secondary antibody (Invitrogen, Waltham, MA, USA) for 1 h in the dark. Nuclei were stained with 4′,6-diamidino-2-phenylindole and observed using an Eclipse Ti-U fluorescent microscope (Nikon, Tokyo, Japan). The fusion index was determined by calculating the ratio of nuclei present in MHC-positive cells with 2 or more nuclei among the total nuclei in the randomly selected region.
10
Fluorescence Imaging of Live Cells
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Live cells were imaged by fluorescence microscopy at the indicated time points using 20X magnification on an Eclipse Ti-U fluorescent microscope (Nikon). Images were acquired with a D5-QilMc digital camera and analyzed using NIS-Elements software (Nikon).
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