Cells grown on various matrices were fixed by 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 in PBS for 10 min, and blocked by 1% BSA in PBST (PBS-0.5% tween 20). The primary antibodies used in this study included rabbit anti-β1-integrin (Santa Cruz Biotechnology, Dallas, TX, USA, 1:100 dilution), rabbit anti-E-cadherin (Abcam, MA, USA, 1:25 dilution), and mouse anti-vimentin (Abcam, MA, 1:200 dilution). Secondary antibodies conjugated with Alexa Fluor 594 and 488 fluorescent dyes were purchased from Invitrogen (Carlsbad, CA, USA) and used at 1:200 dilution. The slides were then subjected to fluorescent imaging using a Nikon TE-U 2000 microscope. For each experiment on a particular marker, imaging parameters, such as the exposure time, gain value, image size, and magnification, were kept constant through the entire study for all samples. Quantitative analyses were performed using ImageJ software (NIH free download). Background signals, measured in the absence of primary antibody, were subtracted for fluorescence intensity analysis.
Te2000 u microscope
Manufactured by Nikon
Sourced in Japan, United States, Germany, Netherlands
The Nikon TE2000-U is an inverted fluorescence microscope designed for advanced live cell imaging and high-resolution microscopy. It features a modular design, allowing for customization to meet specific research requirements. The TE2000-U provides stable and reliable performance for a wide range of applications, including cell biology, developmental biology, and neuroscience research.
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Lab products found in correlation
Fura 2 am, by Thermo Fisher Scientific (9 mentions)
Act 1, by Nikon (9 mentions)
Dxm1200f digital camera, by Nikon (7 mentions)
Lambda 10 2, by Sutter Instruments (5 mentions)
Metamorph 6, by Molecular Devices (5 mentions)
Axiocam mr3, by Zeiss (5 mentions)
Axio observer z 1 sd microscope, by Zeiss (5 mentions)
Orca er, by Hamamatsu Photonics (4 mentions)
L 15 medium, by Thermo Fisher Scientific (4 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Matrigel, by BD (3 mentions)
Dxm1200f digital, by Nikon (3 mentions)
Plan apochromatic 63 na 1.4 objective, by Zeiss (3 mentions)
Epidermal growth factor (egf), by Bio-Techne (3 mentions)
Insulin, by Merck Group (3 mentions)
Hybridisation buffer, by Roche (3 mentions)
Anti dig antibody, by Roche (3 mentions)
Diaminobenzidine dab, by Merck Group (3 mentions)
Image pro plus 6, by Media Cybernetics (3 mentions)
1.45 numerical aperture tirf objective, by Nikon (3 mentions)
186 protocols using te2000 u microscope
1
Immunofluorescence Imaging of Cell Matrices
2
Immunofluorescence Imaging of Cell Matrices
3
Quantifying Extracellular Matrix Proteins
4
Quantifying Extracellular Matrix Proteins
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The cells grown on various matrices were fixed by methanol for 5 min at ‒10 °C followed by air-drying and then blocked with 10% bovine serum albumin (Sigma-Aldrich, St. Louis, MO) in PBS for 30 min. The cells were then incubated with primary antibodies for 60 min at room temperature in a shaker. Primary antibodies used in this study included rabbit anti-collagen I (Abcam, MA, 1:100), mouse anti-collagen III (Abcam, MA, 1:100) and mouse anti-α-SMA (Abcam, MA, 1:200 dilution). Secondary antibodies, purchased from Invitrogen (Carlsbad, CA), were used at 1:200 dilution and incubated with the samples for 1 h in a dark, humidified chamber. The slides were then subject to fluorescence imaging using a Nikon TE-U 2000 microscope. To rule out the influence of dye color in measuring the fluorescence intensity, Invitrogen™ Alexa Fluor™ 488 (Carlsbad, CA) and Invitrogen™ Alexa Fluor™ 594 (Carlsbad, CA) at 1:1000 dilution were used to calibrate the dye intensity. By switching the staining color for COLI and COLIII, we could also confirm the nearly identical binding affinity of the antibodies (< 5%). Quantitative analysis was carried out using ImageJ software. Background signals were subtracted to estimate the fluorescence intensity.
5
Wound Healing Assay with CXCL12 and AMD3100
6
Histological Analysis of Liver Tissue
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For hematoxylin-eosin (H&E) staining, liver paraffin sections were dewaxed and rehydrated, and the sections were stained with H&E. Histological observations were made by light microscopy. For immunohistochemical staining, paraffin sections were dewaxed and hydrated and stained with the indicated primary antibodies. The coloration reaction was performed according to the SABC Kit (SolarBio, Beijing, China) manufacturer's protocols. Images were acquired with a Nikon TE2000-U microscope (Nikon, Kyoto, Japan), and the mean integral optical density (IOD) was analyzed with Image Pro-Plus 6.0 software (Media Cybernetics, Rockville, MD, USA).
7
Fura-2 Based Calcium Imaging in ECs
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Intracellular calcium release in ECs was assessed through microfluorimetric measurements of the cytosolic Ca2+ concentration by using fura-2 as described previously [36 (link)]. In brief, ECs were incubated with 5 μM fura-2 AM (Invitrogen, Carlsbad, CA, USA) for 1 h at 37 °C and subsequently washed and bathed in DMEM supplemented with 10% FBS and penicillin–streptomycin solution (100 units/mL, 100 μg/mL; Invitrogen) under 5% CO2. The cells were alternately excited at 340 and 380 nm using an optical filter changer (Lambda 10-2, Sutter Instruments, Novato, CA, USA). Emission was measured at 500 nm, and images were captured using a charge-coupled device camera (CoolSnap HQ2, Photometrics) attached to an inverted Nikon TE 2000-U microscope. The captured images were analyzed using MAG Biosystems Software. All experiments were performed at room temperature (approximately 25 °C).
8
Cell Migration and Invasion Assays
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HeLa, SiHa, and C33A cells were transfected with the miR-205 mimic, NC, miR-205 inhibitor, or inhibitor NC. Transfected cells were harvested and subjected to the following assays at 48 h after transfection. For migration assays, the transfected cells (0.5 × 106 cells/mL) were seeded in the top of a chamber containing a membrane with 8.0-μm pores (Corning Costar Corp., Cambridge, MA, USA). Following a 12–18 h incubation period, cells that passed through the membrane were fixed and stained with hematoxylin. Cells were scraped and removed from the top of chamber. Membranes were mounted on cover slides, and cells were counted. Cell migration was quantified by counting the number of cells passing through the pores from five different randomly selected fields of view per sample at 100× magnification under a microscope. For invasion assays, Matrigel (BD Biosciences) diluted to 1 mg/mL in serum-free cold cell culture medium was added to the top of a chamber containing a membrane with 8.0-μm pores and incubated at 37 °C overnight until the Matrigel solidified. Analysis was then carried out as described above. Samples were viewed under a Nikon TE 2000-U microscope (Nikon, Tokyo, Japan).
9
EGFR-Grb2 Interaction Dynamics
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Suitable clones of BaF/3 cells
(transfected with EGFR–eGFP or cotransfected with EGFR–eGFP
and mRFP–Grb2) were selected using flow cytometry as described
previously.8 (link) Cells from each clone were
collected by centrifugation (5 mL culture, 1400 rpm, 4 min, 4 °C),
serum starved for 3 h at 37 °C in serum-free medium, and then
resuspended in PBS containing 0.25% BSA and 10 μM phenyl arsine
oxide (to block receptor internalization7 (link)−9 (link)). Half of the cell suspension
was treated with EGF (final concentration: 16 nM) and half with an
equivalent volume of buffer. After 20 min, the cells were aliquoted
onto a coverslip of an inverted chamber (ambient conditions, ca. 23
°C) and imaged with a frequency-domain lifetime-imaging microscope
(100× NA1.2 oil objective, 470 nm LED, FITC filter block, Nikon
TE2000U microscope; Nikon Inc., Japan) coupled to a LIFA lifetime
attachment (Lambert Instruments, The Netherlands). Lifetime images
were corrected for instrument response (pixel-dependent instrument
phase and modulation) with a solution of rhodamine 6G in distilled
water (lifetime: 4.1 ns).31 (link) BaF/3 cells
(nontransfected) were also measured to determine the lifetime characteristics
of cell background fluorescence.
(transfected with EGFR–eGFP or cotransfected with EGFR–eGFP
and mRFP–Grb2) were selected using flow cytometry as described
previously.8 (link) Cells from each clone were
collected by centrifugation (5 mL culture, 1400 rpm, 4 min, 4 °C),
serum starved for 3 h at 37 °C in serum-free medium, and then
resuspended in PBS containing 0.25% BSA and 10 μM phenyl arsine
oxide (to block receptor internalization7 (link)−9 (link)). Half of the cell suspension
was treated with EGF (final concentration: 16 nM) and half with an
equivalent volume of buffer. After 20 min, the cells were aliquoted
onto a coverslip of an inverted chamber (ambient conditions, ca. 23
°C) and imaged with a frequency-domain lifetime-imaging microscope
(100× NA1.2 oil objective, 470 nm LED, FITC filter block, Nikon
TE2000U microscope; Nikon Inc., Japan) coupled to a LIFA lifetime
attachment (Lambert Instruments, The Netherlands). Lifetime images
were corrected for instrument response (pixel-dependent instrument
phase and modulation) with a solution of rhodamine 6G in distilled
water (lifetime: 4.1 ns).31 (link) BaF/3 cells
(nontransfected) were also measured to determine the lifetime characteristics
of cell background fluorescence.
10
Microscopy Imaging Techniques Protocol
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For phase contrast microscopy, a Zeiss Primo Vert microscope (Carl Zeiss, Oberkochen, Germany) and a Nikon TE 2000-U microscope (Nikon, Amsterdam, The Netherlands) with a 10× objective were used. The Nikon microscope used a DXM1200F digital camera with Nikon ACT-1 software (Melville, NY, USA). For experiments where image acquisition was performed using fluorescence, an Axio Observer Z.1 SD microscope (Carl Zeiss, Germany) was used, coupled to an AxioCam MR3, and with the Plan Apochromatic 63×/NA 1.4 objective. The deconvolution was performed with the software AxioVision Release 4.8.2 SPC, and the images were processed using ImageJ version 1.51.
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