S2 cells were grown on 12mm coverslips and fixed in 4% paraformaldehyde for 20 minutes at room temperature. Cells were washed in TBS (20 mM Tris HCl pH 7.6, 150 mM NaCl) and blocked for 30 minutes in blocking buffer (5% normal goat serum in TBS containing 0.1% Triton X-100 (TBST) for 1 hour at room temperature. Incubation with primary antibodies was performed for 2 h at room temperature (for SH3PX1) or overnight at 4°C (for all remaining antibodies) in TBST. Following incubation with Alexa Fluor-conjugated secondary antibodies (Thermo Fisher Scientific) for 2 hours at room temperature, coverslips were mounted in Vectashield mounting medium with DAPI (Vector Laboratories) and visualized on a DeltaVision fluorescence microscope (Applied Precision) using a 60x/ 1.42 Plan Apo oil objective. Images were collected with a Z optical spacing of 0.2 μm using a CoolSNAP HQ (Photometrics) camera with MetaMorph software (Molecular Devices). Predictive deconvolution was then carried out on raw images using Huygens software (Scientific Volume Imaging). Quantification of multi-nucleation was carried out in FIJI (National Institutes of Health). All images were adjusted for contrast using Adobe Photoshop CS6 and figures were assembled using Adobe illustrator CS6.
Deltavision fluorescence microscope
Manufactured by Cytiva
Sourced in United States
The DeltaVision fluorescence microscope is a high-performance imaging system designed for advanced fluorescence microscopy applications. It provides high-resolution, high-contrast imaging capabilities for a wide range of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
Huygens software, by Scientific Volume Imaging (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Lsm 700 confocal microscope, by Zeiss (1 mentions)
Phospho dna pkcs s2056, by Abcam (1 mentions)
Hrp conjugated secondary antibody, by Vector Laboratories (1 mentions)
Cleaved caspase 3 asp175 5a1e, by Cell Signaling Technology (1 mentions)
Rad51 14b4, by GeneTex (1 mentions)
Duolink image tool, by Olink (1 mentions)
Mitotracker green, by Merck Group (1 mentions)
Plan apo 1.4 oil immersion objective, by Olympus (1 mentions)
Softworx 5, by Cytiva (1 mentions)
9 protocols using deltavision fluorescence microscope
1
Immunofluorescence Microscopy of S2 Cells
2
Bmal1 C-terminus Regulates Localization
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To analyze the effect of Bmal1 C-terminal region on the localization of BMAL1 and CLOCK simultaneously, VENUS protein was fused to BMAL1wt and BMAL1GTΔC, and CERULEAN (CERUL) to CLOCK as indicated [24 (link)]. For BiFC assay, amino acid residues 1 to 172 (VN) and 173 to 238 (VC) of VENUS protein was fused to BMAL1 and CLOCK, respectively. NIH-3T3 cells were transfected with the indicated constructs, incubated at 37°C for 36hr and visualized by using Delta-Vision fluorescence microscope (Applied Precision, Isaquah, WA).
3
Immunofluorescence Staining of Cultured Cells
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Cells were seeded at a density of ∼1.0 × 105 cells in coverglass- bottomed dishes (SPL Life Sciences) and incubated for 24 h (26) (link). Cells were fixed with ice-cold 100% methanol for 20 min at −20℃ and washed twice with PBS. Fixed cells were incubated for 1 h at room temperature (RT) in 10% bovine serum diluted in PBS and then with the primary antibodies for 3 h at RT. Alexa Fluor Dye®-conjugated anti-rabbit or anti-mouse IgG (Molecular Probes) were used as the secondary antibodies and were incubated with the cells for 1 h. After the cells were washed, the nuclei were counterstained using 4',6-diamidino-2-phenylindole (DAPI; Invitrogen). Images were obtained using a Delta Vision fluorescence microscope (Applied Precision) and LSM700 confocal microscope (Carl Zeiss).
4
Immunohistochemical Analysis of DNA Damage
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Antigen retrieval was performed using citrate buffer (10 mM, pH 6) or high-pH antigen retrieval solution (Vector). Sections were blocked in 10% goat serum and incubated with antibodies overnight at 4°C followed by fluorophore-conjugated antibody for immunofluorescence or HRP-conjugated secondary antibodies (Vector) for immunohistochemistry. Antibodies used were phospho DNA-PKcs-S2056 (Abcam) and phospho-histone H2AX Ser139 (γH2AX, 20E3, Cell Signaling Technology) on human and mouse tissue; RAD51 (14B4, Genetex), T1α (clone 8.1.1, DHSB), and cleaved caspase 3 Asp175 (5A1E, Cell Signaling Technology) on mouse tissue; and T1α (NC-08, Biolegend) on human tissue. Nuclei were counterstained with DAPI where appropriate. Images were acquired using a DeltaVision fluorescence microscope (Applied Precision).
5
Duolink PLA for EGFR-IGF-1R Interaction
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A Duolink®(Olink Biosciences) in situ proximity ligation assay was performed following the manufacturer’s instructions. Briefly, cells were fixed with ice-cold 100% methanol for 20 min at −20℃ and washed twice with PBS. Fixed cells were incubated in 10% bovine serum in PBS for 1 h at RT and then with the two primary antibodies anti-EGFR rabbit IgG (Santa Cruz) and anti-IGF-1R mouse IgG (Santa Cruz) for 3 h at RT. The secondary antibodies conjugated with oligonucleotide plus or minus probes were diluted with 10% bovine serum in PBS and were added to the cells after they were washed twice in Tris-buffered saline containing Tween 20. The cells were incubated for 1 h at 37℃. After probe hybridization, they were washed twice with wash buffer A supplied in the Duolink kit and then incubated in a solution containing ligase for 30 min at 37℃. After washing, polymerase was added to the amplification solution and the mixture was incubated for 1 h 40 min at 37℃. Cells were washed twice in wash buffer B from the kit and the nuclei were counterstained with DAPI. Images were obtained using a Delta Vision fluorescence microscope (Applied Precision) and analyzed using the Duolink®ImageTool (Olink Biosciences).
6
Fluorescent Protein Imaging Protocol
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For the analysis of fluorescently tagged proteins, cells were washed once with PBS, settled onto glass slides and fixed with 4% paraformaldehyde in PBS for 5 min. Cells were then permeabilized with 0.1% NP-40 in PBS for 5 min and embedded in mounting media (1% w/v 1,4-diazabicyclo[2.2.2]octane, 90% glycerol, 50 mM sodium phosphate pH 8.0) containing 100 ng ml−1 4,6-diamidino-2-phenylindole dihydrochloride (DAPI). Images were captured on a DeltaVision fluorescence microscope (Applied Precision) installed with soft Wo Rx v. 5.5 housed in the Oxford Micron facility. Fluorescent images were captured with a CoolSNAP HQ camera and processed in Image J [52 (link)]. Cell cycle stages of individual cells were estimated as described previously [53 (link),54 (link)].
7
Imaging Apoptosis in FADD-Deficient Jurkat Cells
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FADD-deficient Jurkat cells were seeded onto an 18-mm cover glass in a 12-well tissue culture plate. Cells were loaded with fluorescence probes (150 nM MitoTracker Green and 3 μM propiduim iodide, Sigma-Aldrich) in the presence of 10 ng/ml TNF-α in complete medium for 3 h before time-lapse imaging. Time-lapse imaging was performed on a Deltavision Fluorescence microscope (Applied Precision, Issaquah, WA, USA) equipped with a 37 °C climate chamber with an Olympus (Münster, Germany) × 40 Plan Apo 1.4 oil immersion objective. Images of GREEN were acquired with FITC excitation (490/20) and emission (528/38) filters, and of RED with CY3 excitation (555/28) and emission (617/73) filters. The time interval of imaging was 5 min for a total duration of 6 h starting 3 h after TNF-α treatment.
8
Wide-field Fluorescence Microscopy Protocol
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Cells were imaged using a wide-field DeltaVision fluorescence microscope (Applied Precision) with a 40 × oil immersion objective and Optovar lens (1.6 ×), using softWoRx 5.0.0 software (Applied Precision). AlexaFluor 555 was imaged with 540DF40 excitation, 600DF50 emission, and a Chroma 84100bs polychroic filter set. Typical exposure times were 0.1–0.5 s. Samples shown in the same figure were imaged, analyzed, and displayed under identical conditions.
9
Immunostaining of Larval Fillets
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Larval fillets were immunostained as previously described20 (link). Image stacks were acquired on a Deltavision fluorescence microscope (Applied Precision) at 20× (0.75 NA), and projected. Newly hatched larvae were raised in 500 μl of medium containing 10 μl of 12.4 mM Myriocin in 50 mM NaOH or 1 mM Fumonisin B1 in water.
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