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)).
Csu x1 5000 rpm
Manufactured by Yokogawa
The CSU-X1 5000 rpm is a centrifuge instrument designed for laboratory applications. It is capable of reaching a maximum rotational speed of 5000 rpm. The core function of this product is to separate and isolate different components within a sample through the application of centrifugal force.
Automatically generated - may contain errors
3 protocols using csu x1 5000 rpm
1
Multi-Imaging Techniques for Cell Analysis
2
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.
3
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.
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