Cells were grown on glass coverslips and pulsed with 10 µM EdU and/or 10 µM BrdU for 30 min. For EdU/BrdU kinetics, cells were grown in media containing 5 µM thymidine between the two stainings. Cells were then fixed for 15 min in PBS with 3.2% formaldehyde and permeabilised in 0.2% Triton X-100 for 10 min, then treated with 4 N HCl for 20 min. All cells were washed and blocked (3% BSA, 0.1% Tween 20 in PBS) for 45 min. Click-iT- EdU Alexa 530 staining was performed as per manufacturer instruction (Invitrogen). After three washes in PBS, cells were incubated with monoclonal anti-BrdU antibody (1/400, MoBu-1 Exbio, #11-286-100), and revealed by an Alexa Fluor 633 conjugated goat anti-mouse antibody. Coverslips were mounted using Prolong Gold mounting medium (Invitrogen) and captured using a confocal Leica SP5-SMD microscope, with a Leica ×63/APO 1.4 lens, powered by LASAF software. Serial 0.5 µm Z-sections were taken, and maximum projections were generated by using Image J software.
Sp5 smd microscope
Manufactured by Leica
The Leica SP5-SMD microscope is a confocal laser scanning microscope designed for high-resolution imaging. It features a range of advanced optics and lasers to enable detailed observation and analysis of samples.
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4 protocols using sp5 smd microscope
1
Dual DNA Replication Kinetics Assay
2
Tsg101-Gag Colocalization Reveals Interaction
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To study Tsg101-Gag recruitment by colocalization, HeLa cells were transfected with pCR3.1cherry-Tsg101 (0.2 μg) and pNL4.3MA-YFP WT, ΔZF2 or L− with its respective untagged counterpart pNL4.3Δenv at a molar ratio of 0.2:1:1. Cells were fixed 20 h post-transfection in 4% paraformaldehyde/phosphate buffered saline (PBS) for 20 min at room temperature, washed three times in PBS and imaged with a confocal Leica SP5-SMD microscope equipped with a 63x/1.4 oil immersion objective and Argon-488-nm/DSSP 561-nm lasers. For comparative purposes, exposure times were set equal for all the conditions tested. In addition, Gag–Tsg101 interactions were visualized in situ by using the Duolink technique that reveals whether two proteins might interact in the cell (39 ). HeLa cells were transfected 20 h with cherry-Tsg101 and untagged pNL4-3Δenv WT, ΔZF2 or L- (at the molar ratio 0.2:1). Total Gag and Tsg101 expression in cells was monitored by western blotting (Text S7). Duolink signals were visualized at the PM using TIRFM and fluorescent spots quantified using Image J software. Duolink assays are described in more detail in Text S7.
3
Confocal Imaging of U87 Cells
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Confocal microscopy: 300,000 U87 cells were seeded 24 h before imaging into 35 mm glass bottom dishes (FluoroDish, World Precision Instruments). After the incubation with the WRAP PBN (condition given in the Supplementary Material), live cell images were acquired on an inverted Leica SP5-SMD microscope. Image acquisitions were done sequentially to minimize crosstalk between the fluorophores. Each confocal image was merged and adjusted with the same brightness and contrast parameters using the ImageJ software.
4
Tracking Dynamic Behavior of Dorsal Cells
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Embryos were imaged using Leica SP5-SMD microscope equipped with a motorized stage, as well as a heated-regulated incubator. Nomarski and fluorescent images were taken every 4 min during 30 min, using a LEICA 10x/0.3 HCX PL objective and captured with a hybrid detector (Leica HyD) controlled using the C software, focusing on the internal layers of the DCs at the equatorial/dorsal embryonic axis at 75% epiboly. Stacks of 10 planes with a Z step of 1 µm were taken for each time point. Manual cell tracking and "x/y" cell position recording (of each cell at each time point) were performed using ImageJ software (Manual Tracking plugin) and the cell migration parameters were calculated using Ibidi Chemotaxis tool (http://ibidi.com/software/chemotaxis_and_migration_tool/).
Data of the dynamic behavior of DCs were extracted to quantify the directionality. Time of contact was calculated following pairs of DCs throughout the experiment and extracting their contact time.
For 3-D reconstruction stacks of confocal images were processed with Imaris (Bitplane, Zurich, Switzerland) and volume representations were made. 3-D cell tracking was performed to measure Total Speed, Instantaneous Speed, Net Speed and Persistence.
Data of the dynamic behavior of DCs were extracted to quantify the directionality. Time of contact was calculated following pairs of DCs throughout the experiment and extracting their contact time.
For 3-D reconstruction stacks of confocal images were processed with Imaris (Bitplane, Zurich, Switzerland) and volume representations were made. 3-D cell tracking was performed to measure Total Speed, Instantaneous Speed, Net Speed and Persistence.
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