Cells were seeded in a 4-well Lab-Tek Chamber slide (ThermoFisher Scientific). At given time points, transfected cells (with or without treatment) were washed three times with phosphate-buffered saline (PBS), then fixed in PBS containing 4% paraformaldehyde (Sigma-Aldrich) at room temperature for 20 min, and permeabilized with 1% Triton X-100 (Sigma-Aldrich) in PBS at room temperature for 20 min. After three washes, cells were treated with PBS containing 2% FBS for blocking at room temperature for 1 h, followed by 1 h incubation with primary antibody and secondary antibody (anti-mouse or anti-rabbit IgG conjugated with Alexa Fluor 488 or 568). Cells were washed for three times, and counterstained with 4’,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA). Images were obtained by using Eclipse Ti2 inverted fluorescence microscope (Nikon).
Eclipse ti2 inverted fluorescence microscope
Manufactured by Nikon
Sourced in Japan
The Eclipse Ti2 is an inverted fluorescence microscope designed for advanced imaging applications. It features a large working distance and a wide range of objectives to accommodate a variety of sample types. The Eclipse Ti2 is equipped with high-performance optics and a sensitive camera system to capture detailed fluorescence images.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield mounting medium with dapi, by Vector Laboratories (2 mentions)
Lab tek 2 chamber slide, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Merck Group (1 mentions)
Lab tek chamber slide, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
5 protocols using eclipse ti2 inverted fluorescence microscope
1
Immunofluorescence Microscopy of Transfected Cells
2
Quantifying Smooth Muscle Cell Response
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VSMCs cultured in 60 mm dishes were pretreated with NE or DO-NE for 24 h.
Thereafter, the cells were exposed to vehicle (Dimethyl sulfoxide, DMSO) or Ang
II for 72 h, washed briefly with cold phosphate-buffered saline, and then
stained with a solution for 24 h at 37°C as described previously (Kim et al., 2012 (link)). Fluorescence microscopy
was performed to detect stained cells. The number of stained cells in four
individual captured images was counted on a computer monitor using a DS-Ri2
camera equipped to Nikon Eclipse Ti2 inverted fluorescence microscope (Tokyo,
Japan) by an independent observer.
Thereafter, the cells were exposed to vehicle (Dimethyl sulfoxide, DMSO) or Ang
II for 72 h, washed briefly with cold phosphate-buffered saline, and then
stained with a solution for 24 h at 37°C as described previously (Kim et al., 2012 (link)). Fluorescence microscopy
was performed to detect stained cells. The number of stained cells in four
individual captured images was counted on a computer monitor using a DS-Ri2
camera equipped to Nikon Eclipse Ti2 inverted fluorescence microscope (Tokyo,
Japan) by an independent observer.
3
Soft Agar Colony Formation Assay
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The soft agar colony formation assay was performed as previously described (17 (link), 18 (link)). Briefly, 100, 200, 1 × 103, or 1 × 104 cells were suspended in 2 ml of 0.6% top agar (Sigma–Aldrich) and plated onto 1.2% base agar in 6-well plates and irradiated with a 0-, 2-, 4-, or a 6-Gy dose of 160 kV X-rays (RAD SOURCE, USA). The irradiated cells were cultured for 14 days. Colonies with a diameter of >50 µm were counted and imaged at ×4 magnification using a Nikon ECLIPSE Ti2 inverted fluorescence microscope. The cloning efficiency was calculated by dividing the number of colonies by the number of cells plated. Each measurement was the average ± standard deviation (SD) of three experiments.
4
Immunofluorescence Imaging of Transfected Vero Cells
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8 × 104 Vero Cells were seeded into each well of an 8-well Lab-Tek II chamber slide (Thermo Fisher Scientific). The next day, cells were transfected with 0.5 μg of DNA per well. At selected time points, cells were fixed with chilled methanol at −20 °C for 30 min. After 1 h incubation in blocking buffer (PBS supplemented with 1% FBS and 0.05% Tween-20), cells were incubated with the primary antibody 4G2 for 1 h. After three PBS washes, cells were incubated with goat anti-mouse IgG conjugated with Alexa Fluor 568 (1:1000 diluted in blocking buffer) for 1 h. Finally, after three PBS washes, cells were mounted in a Vectashield mounting medium with DAPI (Vector Laboratories). Fluorescence images were acquired under Eclipse Ti2 inverted fluorescence microscope (Nikon Instruments Inc.).
5
Immunofluorescence Staining of Cultured Cells
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