MDCK cells were fixed in 2% glutaraldehyde in PBS for 1 h, washed, and gradually equilibrated to 30% glycerol as cryoprotectant. The cells were lifted with a cell scraper and rapidly frozen by contact with a polished gold block cooled to −186°C using a LifeCell (Bridgewater, NJ) CF-100 device. Freeze fracture of the samples was performed with a Balzers (Balzers, Liechtenstein) freeze fracture/etch apparatus at −110°C, and samples were unidirectionally shadowed at 45° with platinum and stabilized with carbon deposited from 90°. Replicas were cleaned with sodium hypochlorite and collected onto copper TEM grids. Transmission electron microscopy of the replicas was performed using a JEOL 2100 TEM operating at 200 kV with an Orius 832 camera (Gatan, Pleasanton, CA). Data collection and analysis were performed using the SerialEM/Etomo software suite (Mastronarde, 2005 (link)). Average strand number was defined as the number of strands across the tight junction at every 500-nm interval; n = 40; five pairs of cells were used for each wild type and knockout.
Orius 832 camera
Manufactured by Ametek
Sourced in United States
The Orius 832 is a high-resolution digital camera designed for scientific and industrial applications. It features a 3.2-megapixel CCD sensor and can capture images at a resolution of 2048 x 1536 pixels. The camera is capable of operating in a wide range of exposure times, from 1 microsecond to 1 second, making it suitable for various imaging requirements.
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3 protocols using orius 832 camera
1
Freeze Fracture and Electron Microscopy of Tight Junctions
2
High-resolution Imaging with TEM
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TEM creates images with a higher resolution than a light microscope by transmitting electrons. With this technique, the smallest details of a particle can be seen. TEM images were obtained using a CM120 electron microscope (Philips, USA) equipped with a Tietz 2K × 2K CCD camera and a fiber optically coupled Gatan Orius 832 camera.
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TEM creates images with a higher resolution than a light microscope by transmitting electrons. With this technique, the smallest details of a particle can be seen. TEM images were obtained using a CM120 electron microscope (Philips, USA) equipped with a Tietz 2K × 2K CCD camera and a fiber optically coupled Gatan Orius 832 camera.
3
Visualizing the HP1γ Dimer Structure
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For visualizing the shape and contour of the HP1γ dimer, we produced and purified an N-terminal 6×His-tagged recombinant form of this protein using the pET vector system (Novagen, CA). The HP1γ-encoding plasmid was grown in DE3 BL21 bacteria cells overnight and induced with 0.5 mM IPTG for 90 min at 32 °C. The recombinant protein was purified using the Thermo Scientific HisPur Cobalt Resin Kit according to the manufacturer’s instructions. Protein was dialyzed overnight and concentrated to a final concentration of 1 mg/ml. For visualization at the electron microscopy level, 10 μl of the purified protein solution was placed on the surface of glow-discharged formvar carbon-coated grids. After 30 s, the grids were blotted and stained for 30 s in 1 % uranyl acetate. Micrographs were acquired using a JEOL, JEM-1400Plus TEM at 80-kV accelerating voltage, equipped with a Gatan Orius 832 camera.
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