Immunostaining was performed as described previously78 (link) and visualized using an Axiovert 200MAT microscope (Carl Zeiss) and an A1R-MP confocal laser-scanning microscope (Nikon). For time-lapse recording, EGFP-ATP11C expressing HeLa cells were placed on a microscope stage prewarmed to 37 °C, and then observed on an A1R-MP confocal microscope. After 5 min recording, PMA was administered, and images were recorded every 7.8 s for a total of 34 min (Supplementary Movie 1 ). For Supplementary Movie 2 , the cells were incubated in serum-free medium containing 2% BSA for 3 h. After 5 min recording, 5-HT was administered and images were recorded every 8.0 s for a total 79 min.
Axiovert 200mat microscope
Manufactured by Zeiss
Sourced in Germany
The Axiovert 200MAT is an inverted microscope designed for materials analysis. It features fully motorized components for automated operation and high-precision control of the sample position. The microscope is equipped with a range of illumination options and objectives to support various imaging techniques, including brightfield, darkfield, and polarized light microscopy.
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4 protocols using axiovert 200mat microscope
1
Visualizing EGFP-ATP11C Dynamics in HeLa Cells
2
Imaging HeLa Cell Protein Localization
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HeLa cells were cultured in minimal essential medium (Nacalai Tesque) supplemented with 10% heat-inactivated fetal bovine serum at 37°C under a 5% CO2 atmosphere. HeLa cells were transfected with expression plasmids for CDC50A and ATP10 proteins, ATP11 proteins, or their chimeric constructs using FuGENE6 (Promega), and incubated for 48 h. HeLa cells were transfected with Lyn11-EGFP–fused constructs or HA-tagged ATP10B-NT and ATP11B-CT constructs using FuGENE6 (Promega), and incubated for 24 h. Immunostaining was performed as described previously (Shin et al., 2004 (link); Takatsu et al., 2014 (link)) and was visualized using an Axiovert 200MAT microscope (Carl Zeiss) and an A1R-MP confocal laser scanning microscope (Nikon). Briefly, HeLa cells were fixed with 3% paraformaldehyde and then permeabilized with 0.2% saponin for 20 min or 0.1% Triton X-100 for 5 min at room temperature. The cells were blocked with 10% fetal calf serum in phosphate-buffered saline (PBS) at room temperature for 30 min and incubated sequentially with primary and secondary antibodies at room temperature for 1 h. Coverslips were placed using Mowiol mounting medium.
3
Microstructural Analysis of Metal Parts
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In order to make microstructural analysis possible, the authors extracted cut-outs from the studied parts according to the plan shown on Figure 5 . The samples from the scraper shovel and the connector chain linkage were cut and grinded in such a manner, that they had two smooth and parallel planes, as is shown on Figure 6 . The samples were enclosed in a resin scaffold and polished. The polishing process included sanding with a sandpaper of different grits (120–1200 grit) and finishing polish performed on a Struers Pedemax-2 polishing machine (Struers, Ballerup, Denmark) with an Al2O3 suspension in order to obtain a mirror-like surface.
The prepared surfaces of the samples cross-sections were etched with 6% nital. The microstructure at such conditions was examined using a Carl Zeiss Axiovert 200 MAT microscope (Carl Zeiss Microscopy Deutschland GmbH, Oberkochen, Germany). Average grain size for samples SS1 and SS2 (unloaded) was determined using the Jeffries planimetric method with Saltykov rectangle correction.
The prepared surfaces of the samples cross-sections were etched with 6% nital. The microstructure at such conditions was examined using a Carl Zeiss Axiovert 200 MAT microscope (Carl Zeiss Microscopy Deutschland GmbH, Oberkochen, Germany). Average grain size for samples SS1 and SS2 (unloaded) was determined using the Jeffries planimetric method with Saltykov rectangle correction.
4
Immunostaining and Polarization Analysis
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Immunostaining was performed as previously described (Shin et al., 2004; (link)Takayama et al., 2019) (link) and visualized using an Axiovert 200MAT microscope (Carl Zeiss). MDA-MB-231 cells seeded onto coverslips were fixed with 3% paraformaldehyde. To preserve the polarity of Ba/F3 cells, 3% PFA was added directly to cultured cells and incubated at room temperature for 30 min. Cells were then permeabilized with 0.2% saponin for 30 min or 0.1% Triton X-100 for 5 min at room temperature. We evaluated the polarized localization of pERM in Ba/F3 cells by both visual assessment and line-scan using the ZEN software (Carl Zeiss) to measure fluorescence intensities across the backs and fronts of cells. The ratio of the peak fluorescence intensity of the back to front was estimated. Polarized localization of ezrin(WT) and ezrin(T567D) mutant was also estimated by the ratio of the peak fluorescence intensity of the back to front. The fluorescence intensities of MPAct and phalloidin across the backs and fronts of Ba/F3 cells were analyzed by line-scan.
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