The experimental approach for immunofluorescence microscopy are adapted from previous literature (64 (link)). Briefly, HEK293 cells were cultured on poly-lysine coated coverslips, transfected with plasmids expressing FLAG-tagged HO2 and its variants, washed with phosphate buffered saline (PBS), and fixed with 4% w/v paraformaldehyde before immunostaining. Immunofluorescence microscopy samples were permeabilized with 1× PBS containing 0.2% v/v Triton X-100 and stained with corresponding antibodies. Sample images were acquired on an Olympus IX81 microscope with a Yokogawa CSU-W1 spinning disk confocal scanner unit, an Olympus PlanApo 60× 1.42 NA objective, and a Hamamatsu ImagEMX2-1K EM-CCD digital camera.
Ix81 microscope
Manufactured by Yokogawa
Sourced in United Kingdom, Sweden
The IX81 microscope is a high-performance inverted research microscope designed for a variety of applications. It features a modular design and supports a range of imaging techniques, including brightfield, phase contrast, and fluorescence microscopy. The IX81 provides a stable and versatile platform for researchers and laboratories.
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Lab products found in correlation
Csu x1 5000 rpm, by Yokogawa (3 mentions)
Ixon3 du 885, by Oxford Instruments (3 mentions)
Csu x1 spinning disc, by Yokogawa (3 mentions)
Imagemx2 1k em ccd digital camera, by Hamamatsu Photonics (2 mentions)
Tcs sp5, by Leica (2 mentions)
Fv1000, by Leica (2 mentions)
Glass bottom dishes, by MatTek (2 mentions)
Csu x1 spinning disk, by Yokogawa (2 mentions)
Emccd andor du 897 camera, by Oxford Instruments (2 mentions)
Plan apochromat, by Zeiss (1 mentions)
Eclipse ti u inverted microscope, by Oxford Instruments (1 mentions)
Cfi plan apochromat lambda, by Nikon (1 mentions)
Observer d1 inverted microscope, by Zeiss (1 mentions)
Observer z1 microscope, by Zeiss (1 mentions)
Fv1000, by Olympus (1 mentions)
Ix71 microscope, by Olympus (1 mentions)
Csu w1, by Olympus (1 mentions)
Visifrap module, by Visitron (1 mentions)
Visiview, by Visitron (1 mentions)
Inverted wide field microscope, by Zeiss (1 mentions)
22 protocols using ix81 microscope
1
Immunofluorescence Imaging of HO2 Variants
2
Embryo Imaging and Microscopy Protocol
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Gravid hermaphrodites were dissected in osmotically balanced blastomere culture medium (Shelton and Bowerman, 1996 (link)) and the extracted embryos mounted on a 2% agarose pad. Dual fluorescence and DIC time lapse microscopy was performed at room temperature with a 60x CFI Plan Apochromat Lambda (NA 1.4) objective on a Nikon Eclipse Ti-U Inverted Microscope connected to an Andor Zyla 4.2 sCMOS camera, or with a 63x Plan-Apochromat (NA 1.4) objective on a Zeiss ObserverD.1 inverted microscope connected to the same type of camera. One frame was captured every 10 s, and a z-stack was acquired at every time point, covering 20 µm, with a distance of 0.7 µm between focal planes. Time lapse images in Figure 5A,C,J,K ; Figure 3—figure supplement 1E ; Figure 5—figure supplement 1B were acquired at 23°C using an inverted Olympus IX 81 microscope equipped with a Yokogawa spinning disk CSU - W1 with a 63x (NA 1.42 U PLAN S APO) objective and a 16-bit PCO Edge sCMOS camera. Images were obtained using a 488-nm solid-state laser at 60% laser power, with an exposure time of 400 ms. Embryos were imaged every 5 s, and a z-stack was acquired at every time point, covering 2 µm of the embryo cortex, with a distance of 0.5 µm between focal planes (i.e. four cortical planes).
3
Visualizing Cell Cycle Dynamics with TIRF Microscopy
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Live microscopy used an Olympus IX81 microscope equipped with a Yokogawa CSU-X1 scan head, an EM-CCD Cascade II (Photometrics), a ASI MS-2000 Z-piezo stage, and a PlanApo 100×, 1.45 NA total internal reflection fluorescence microscope oil objective. Fluorophores were excited at 561 nm (mCherry, ∼30 μW) and 491 (GFP, ∼75 μW). Bright-field images were acquired using a CoolLED diode. Imaging was performed in filtered, nonautoclaved YPAD using an Onix CellAsic microfluidic chamber to regulate cell synchronization in α factor and the release into 200 mM HU. Time-lapse series (150 min total) of 50 optical slices per stack were acquired for 30 time points at intervals of 5 min, with each slice being exposed for 30 msec per laser line. Images were deconvolved using Huygens Pro and channel-aligned. Lasers were used for the 561 and 491 lines at 6% and 5%, respectively. Bleaching did not occur in these conditions until 5 h.
4
Live Imaging of pHluoflashes in Tissues
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Live imaging was performed with an Olympus IX81 microscope equipped with a spinning disk (CSU-X1 5000 rpm, Yokogawa) and Okolab environmental chamber maintained at 37°C. Image were acquired with a 20X objective by EMCCD camera (iXon3 DU-885, Andor technology). 15-30 planes spaced of 0,5-3µm were imaged for each open-book at 30-minute interval for 10 hours approximatively. To reduce exposure time and laser intensity, acquisitions were done using binning 2x2. Images were acquired using IQ3 software using multi-position and Z stack protocols. Z stack projections of the movies were analyzed in ImageJ software. The analysis of pHluoflashes was performed from time-lapse acquisitions. In some experiments, the time interval was reduced for faster image acquisition. Time intervals of 3 minutes, 5 minutes, 8 minutes and 12 minutes were tested. At 3 and 5 minutes, the tissues were rapidly damaged. We thus selected time interval of 8 min as the better compromise between time resolution and phototoxicity.
Confocal imaging was performed with either an Olympus FV1000 with a 40x objective and zoom or a Leica TCS SP5 with a 63x objective. Deconvolution was done using the Huygens software. 3D surface reconstructions were done using the Imaris software.
Confocal imaging was performed with either an Olympus FV1000 with a 40x objective and zoom or a Leica TCS SP5 with a 63x objective. Deconvolution was done using the Huygens software. 3D surface reconstructions were done using the Imaris software.
5
Multi-Imaging Techniques for Cell Analysis
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Pictures of in situ hybridization experiments were taken using a Z1 observer microscope (Zeiss). Pictures of the apical surface or brain slices were taken with an inverted confocal laser-scanning microscope FV1000 (Olympus). En face live cell imaging was performed using the Olympus IX81 microscope equipped with a spinning disk (CSU-X1 5000 rpm, Yokogawa), an Okolab environmental chamber, an electron multiplying charged-coupled device (EMCCD) camera (iXon3 DU-885), and applied software (Andor Technology). Photographs were analyzed with Fiji software (69). Data collection and analysis were performed blindly. Analysis of cell division orientation was performed as described in (12 (link)). Quantifications are represented as means ± SEM, and sample size and statistical significance are indicated in the figure legends. Statistic tests and graphs were performed with GraphPad Prism software. *P < 0.05 was considered as significant, **P < 0.01, and ***P < 0.001. Measures of the endfeet area were performed with Cell Profiler software as in (60 (link)).
6
Live Imaging of Cellular Dynamics
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Live imaging was performed with an Olympus IX81 microscope equipped with a spinning disk (CSU-X1 5000 rpm, Yokogawa) and Okolab environmental chamber maintained at 37°C. Image was acquired with a 20× objective by EMCCD camera (iXon3 DU-885, Andor technology). Fifteen to thirty planes spaced of 0.5–3 μm were imaged for each open-book at 30 min interval for 10 hr approximately. To reduce exposure time and laser intensity, acquisitions were done using binning 2 × 2. Images were acquired using IQ3 software using multi-position and Z stack protocols. Z stack projections of the movies were analyzed in ImageJ software. The analysis of pHluo-flashes was performed from time-lapse acquisitions. In some experiments, the time interval was reduced for faster image acquisition. Time intervals of 3, 5, 8, and 12 min were tested. At 3 and 5 min, the tissues were rapidly damaged. We thus selected time interval of 8 min as the better compromise between time resolution and phototoxicity. Confocal imaging was performed with either an Olympus FV1000 with a 40× objective and zoom or a Leica TCS SP5 with a 63× objective. Deconvolution was done using the Huygens software. 3D surface reconstructions were done using the Imaris software.
7
Conditional Gβ-Yck1 BiFC Assay
8
Live Embryo Imaging and PCM Disassembly
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For live embryo imaging, we used an inverted Olympus IX81 microscope with a Yokogawa spinning-disk confocal head (CSU-X1), a 60×1.2 NA Plan Apochromat water objective, and an iXon EM+DU-897 BV back illuminated EMCCD camera (Andor Technologies, Belfast, UK). For analysis of PCM disassembly in vivo, we generated 36×0.5 µm Z-stacks every 10 s using 50 ms exposure and 8% laser intensity (4.5 mW; 488 nm laser). In vitro SPD-5 condensates and fixed embryos were visualized with an inverted Olympus IX71 microscope using 60×1.42 NA or 100×1.4 NA Plan Apochromat oil objectives, CoolSNAP HQ camera (Photometrics Tuscon, AZ, USA), and DeltaVision control unit (GE Healthcare, Salt Lake City, UT, USA).
9
Imaging Microtubule Dynamics and Mitochondria in Hippocampal Neurons
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Hippocampal cultures were plated in glass-bottom dishes (MatTek), transduced with virus expressing shRNA, transfected with either EB3-Tomato or Mito-DsRed reporters at 3/4 DIV and imaged 24 h later. For analysis after expression of EGFP-CDK5RAP2 51–100, cultures were co-transfected with plasmid encoding EGFP or fragment EGFP-CDK5RAP2 51–100 together with EB3-Tomato reporter (2:1 ratio) at 3 DIV and imaged 24 h later. For comet analysis in more mature neurons, cultures were co-transfected with shRNAs together with EB3-Tomato reporter (2:1 ratio) at 8 DIV and imaged 48 h later. Live-imaging of EB3-comets and of mitochondria was performed in the soma and/or within the proximal axons of random transfected cells, using an Olympus IX81 microscope equipped with Yokogawa CSU-X1 spinning disc and a temperature controlled CO2 incubation chamber. Image stacks were acquired with 100 × /1.4 OIL immersion objective and an iXon EMCCD Andor DU-897 camera, using iQ2 software. Fluorescent images with pixel size of 0.14 μm were taken at 1 s intervals during 2.5 min for EB3-comets in axons, at 0.5 s intervals during 25 s for EB3-comets in somas, and at 3 s intervals during 6 min for axonal mitochondria.
10
Immunofluorescence protocol for HEK293 cells
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The experimental approach for IF are adapted from previous literature (64) . Briefly, HEK293 cells were cultured on poly-lysine coated coverslips, transfected with plasmids expressing FLAG-tagged HO2 and its variants, washed with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde (w/v) before immunostaining. IF samples were permeabilized with 1x PBS containing 0.2% Triton X-100 (v/v) and stained with corresponding antibodies. Sample images were acquired on an Olympus IX81 microscope with a Yokogawa CSU-W1 spinning disk confocal scanner unit, an Olympus PlanApo 60x 1.42 NA objective, and a Hamamatsu ImagEMX2-1K EM-CCD digital camera.
Supporting Information. This article contains supporting information.
Supporting Information. This article contains supporting information.
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