For live-cell imaging, we replaced the growth medium with FluoroBrite DMEM Media containing 10% FBS, 1% penicillin/streptomycin, and GlutaMax before imaging. Widefield images of mitotic cells were taken on an IX-81 stand (Olympus). The microscope was equipped as described previously (Wu et al., 2016 (link)). The cells were kept at 37°C with a stage top incubator (INUBH-ZILCS-F1; Tokai Hit) in 5% CO2. Cells were optically sectioned using a 500-nm Z step, spanning a 5.0-µm Z depth in total. 50-ms exposure times were used to acquire each plane in all channels.
Ix 81 stand
Manufactured by Olympus
Sourced in Germany
The IX-81 stand is a versatile and advanced microscope platform designed for a wide range of laboratory applications. It provides a stable and reliable foundation for various microscope components, enabling precise and consistent observations. The IX-81 stand is engineered to accommodate a variety of microscopy techniques and accessories, making it a valuable tool for research, analysis, and scientific investigations.
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4 protocols using ix 81 stand
1
Widefield Live-Cell Imaging of Mitosis
2
Acute Hippocampal Slice Activity Dynamics
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Arc-PBS X PCP-GFP animals injected with AAV5-CaMKII-mcherry, were sacrificed 3-weeks post injection and acute hippocampal slices were prepared. The slices were briefly depolarized with 90 mM KCl for 3 mins and returned to ACSF at room temperature (RT). After 90 mins, slices were grouped into three treatment conditions: first group in ACSF, second group was incubated with CHX (100mg/ml) for 1 hour, and in the third group, CHX was added for 1 hour followed by washout. The first two groups were fixed at 2.5 hours, and the group with CHXwashout was fixed 45 min later to allow complete washout. Fixation was done with 4% PFA in PBS overnight, and washed thrice with PBS, and then mounted onto slides with Prolong Diamond (Invitrogen). Imaging was performed on a wide-field fluorescence microscope built around an IX-81 stand (Olympus) and illumination was with 488nm laser, captured on EMCCD camera (Andor, iXon3 DU-897E-CS0-#BV). 300nm z-stacks were acquired, and max-projected to obtain the images of GCs.
3
Measuring Fluorescence Recovery in Myoblasts
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Immortalized myoblasts were grown on coverslips 24–48 h prior to imaging at upwards of 50% confluency at the time of imaging. Coverslips were mounted on holders and washed in Dulbecco’s PBS (DPBS) and cells were imaged live using a 488-nm (for GFP) or 561-nm (for mCherry) lasers attached to the Hamamatsu spinning sisc confocal microscope on an Olympus IX81 stand. Cells were photobleached using the imaging laser and fluorescence recovery was monitored over time. Results were analyzed using Slidebook 6.0. The data for each condition were recorded, and recovery kinetics were analyzed and plotted.
4
Imaging and Quantifying Plant Seedling Morphology
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Images of seedlings exhibiting phloroglucinol staining were taken on a Leica MZ16 stereo microscope equipped with a Leica DFC320 digital camera. Images of hypocotyl and root length were taken on a Leica SAPO stereo microscope equipped with a Nikon Coolpix B500 camera. Aniline blue-stained cotyledons and root cell bulging were imaged with an Olympus FV1000 setup using an inverted IX81 stand and FluoView software (FV10-ASW version 01.04.00.09) (Olympus Europa GmbH, Hamburg, Germany) equipped with a 10x objective (NA 0.3). For assessing cell bulging a projection of a 5 μm z-stack encompassing seven individual optical sections was used. Aniline blue fluorescence was excited at 405 nm using a diode laser and detected at 425 to 525 nm. H2DCFDA and EGFP fluorescence excitation was done at 488 nm using a multi-line argon laser and detected at 502 to 536 nm. For the direct comparisons of fluorescence intensities, laser, pinhole and gain settings of the confocal microscope were kept identical when capturing the images from the seedlings of different treatments. Scan speeds were set at 400 Hz and line averages at between 2 and 4. Measurements on digital micrographs were done using ImageJ software [84 (link)]. Images were adjusted for color and contrast using Adobe Photoshop 2020 software (Adobe, San Jose, USA).
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