HEK293T cells were plated on 35 mm glass-bottomed microwell petri dishes (MatTek Corporation, Ashland, MA) at a confluence of 2.5 × 105. Then they were transfected with 1.5 μg GFP-VANGL1 (WT or Mutant) or 1.5 μg mCherry-FZD6 (WT or Mutant) using Lipofectamine 2000 and cultured for 24 h at 37 °C in an incubator supplied with 5% CO2. Then the culture dish was placed into a temperature-controlled stage (Tokai Hit, Shizuoka-ken, Japan) pre-warmed at 37 °C and supplied with 5% CO2. Images were acquired every 10 min for a total time of 2 h per condition using a CFI Plan Apochromat Lambda 20 ×, NA = 0.45, mounted on a Nikon Eclipse Ti2-E with Yokogawa W1 Spinning Disk Confocal Microscope and Photometrics Prime 95B sCMOS camera with NIS-Elements AR software. Then cell migration analyses were performed using a manual tracking method available in Fiji (ImageJ) software. Graphs were developed using Graphpad Prism software. All values are represented as the standard error of the mean (SEM). Statistical significance was determined using Student’s t test, and p values of less than 0.05 were considered statistically significant.
Eclipse ti2 e
Manufactured by Yokogawa
The Eclipse Ti2-E is a high-performance inverted microscope designed for advanced imaging applications. Its core function is to provide a stable and precise platform for capturing high-quality images and videos of samples under various magnification and illumination conditions.
Automatically generated - may contain errors
Lab products found in correlation
Temperature controlled stage, by Tokai Hit (1 mentions)
Opal650 conjugated tyramide, by PerkinElmer (1 mentions)
Cyanine3 (cy3), by PerkinElmer (1 mentions)
Rnascope multiplex fluorescent detection kit v2, by Advanced Cell Diagnostics (1 mentions)
Tie epifluorescence microscope, by Hamamatsu Photonics (1 mentions)
Orca flash 4, by Hamamatsu Photonics (1 mentions)
Csu w2, by Yokogawa (1 mentions)
Nis element, by Nikon (1 mentions)
Fluorodish plate, by World Precision Instruments (1 mentions)
Lsm880 laser, by Zeiss (1 mentions)
Csu w1, by Zeiss (1 mentions)
4 protocols using eclipse ti2 e
1
Tracking Cell Migration Dynamics
2
In Situ RNA Hybridization of Murine Spleen
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Spleens were fixed in 10% formalin for 24 h at room temperature and embedded in paraffin. In situ RNA hybridization was performed using the RNAscope Multiplex Fluorescent Detection Kit v2 (Advanced Cell Diagnostics) with the following target probes: Mm-Oas3 (catalog no. 1054261-C2), Mm-Ifit3 (catalog no. 508251-C2), Mm-Cd3e (catalog no. 314721-C3), using a previously described protocol84 (link). After the final amplification step, hybridized probes were visualized using Cy3 or Opal650 conjugated tyramide (Perkin Elmer). Sections incubated with a negative control probe targeting the DapB gene from Bacillus subtilis were analyzed in parallel. Positive control probes against murine Ppib and Ubc were used to confirm RNA integrity in each detection channel for each of the analyzed spleens. Images were acquired with a NIKON Eclipse Ti2-E/Yokogawa CSU-W1 confocal spinning disk microscope with a CFI PlanApo λ ×20 objective/0.75 numerical aperture/1 mm working distance and a 50-µm pinhole disc.
3
Widefield and Confocal Microscopy Protocols
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For widefield visualizations, we used a Nikon TiE epifluorescence microscope equipped with a Hamamatsu Orca Flash 4 camera and an oil immersion 100× Plan Apo numerical aperture (NA) 1.45 objective.
For all time-lapses and mammalian cell visualizations, we used a Nikon Eclipse Ti2-E inverted microscope coupled with a Yokogawa CSU W2 confocal spinning disk unit and equipped with a Prime 95B scientific complementary metal oxide semiconductor (sCMOS) camera (Photometrics). For time-lapses, we used a 40× objective with an NA of 1.15 to acquire z-stacks with 2-μm intervals over 6 μm. Each plane was acquired at low laser power for 200 ms, allowing us to threshold out free bacteria in flow from bound bacteria. For stained mammalian cell visualizations, we used a 100× oil immersion objective with an NA of 1.45 to acquire z-stacks with 0.5-μm intervals.
We used NIS Elements (Nikon) for three-dimensional rendering of z-stack pictures.
For all time-lapses and mammalian cell visualizations, we used a Nikon Eclipse Ti2-E inverted microscope coupled with a Yokogawa CSU W2 confocal spinning disk unit and equipped with a Prime 95B scientific complementary metal oxide semiconductor (sCMOS) camera (Photometrics). For time-lapses, we used a 40× objective with an NA of 1.15 to acquire z-stacks with 2-μm intervals over 6 μm. Each plane was acquired at low laser power for 200 ms, allowing us to threshold out free bacteria in flow from bound bacteria. For stained mammalian cell visualizations, we used a 100× oil immersion objective with an NA of 1.45 to acquire z-stacks with 0.5-μm intervals.
We used NIS Elements (Nikon) for three-dimensional rendering of z-stack pictures.
4
Imaging Larval and Juvenile Fish Fins
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Larval and juvenile fish were anesthetized with 74 µg/ml tricaine (MS-222, Syndel) in fish facility system water. Fish or dissected fins were transferred immediately to a 35 mm glass bottom FluoroDish plate (World Precision Instruments). Two or three drops of 1% low-melt agarose, stored at 38°C and cooled before application, were placed on the caudal fin. Fins were quickly flattened to the FluoroDish with a single-hair paintbrush before the agarose hardened.
The following microscopes were used: Nikon Eclipse Ti-E widefield and Nikon Eclipse Ti2-E with Yokogawa CSU-W1 spinning disk attachments, and Zeiss LSM 880 laser scanning confocal microscope. Confocal image stacks were processed using Imaris software to generate single optical slice digital sections, surface renderings, and 3D reconstructions. Adobe Photoshop was used to adjust levels with identical image acquisition and processing settings for a given experiment. Live fish promptly were euthanized or returned to tanks after imaging.
The following microscopes were used: Nikon Eclipse Ti-E widefield and Nikon Eclipse Ti2-E with Yokogawa CSU-W1 spinning disk attachments, and Zeiss LSM 880 laser scanning confocal microscope. Confocal image stacks were processed using Imaris software to generate single optical slice digital sections, surface renderings, and 3D reconstructions. Adobe Photoshop was used to adjust levels with identical image acquisition and processing settings for a given experiment. Live fish promptly were euthanized or returned to tanks after imaging.
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