The DNA template for CpMgm101 binding was a 738-bp EcoRI fragment derived from the mitochondrial telomere of the C. parapsilosis linear mitochondrial DNA. The fragment was prepared by digestion of the pTZ19R-E1 plasmid (2 (link)) with EcoRI followed by a 1 min incubation with exonuclease III to generate single-stranded overhangs at the 5′ termini. CpMgm101 and this DNA were mixed in a 10:1 ratio (w/w) and incubated for 10 min at 30°C in a buffer B. Immediately after incubation they were diluted in spraying buffer (100 mM ammonium acetate, 30% (v/v) glycerol, pH adjusted to 8.0 with NaOH) to a final concentration of 225 μg/ml CpMgm101 and 20 μg/ml DNA. The diluted samples were sprayed onto freshly cleaved mica chips and transferred into a BalTec MED020 high vacuum evaporator equipped with electron guns. The rotating samples were coated with 0.7 nm platinum at an angle of 7°, followed by 7 nm carbon at 45°. The produced replicas were floated off from the mica chips and picked up with 400 mesh Cu/Pd grids. The grids were inspected in an FEI Morgagni 268D TEM operated at 80 kV. Images were acquired using an 11 megapixel Olympus-SIS Morada CCD camera.
Sis morada ccd camera
Manufactured by Olympus
Sourced in Germany
The SIS Morada CCD camera is a scientific imaging device designed for high-quality image capture in various laboratory applications. It features a charge-coupled device (CCD) sensor that provides reliable and accurate image data. The camera's core function is to capture and digitize optical signals for further analysis and documentation.
Automatically generated - may contain errors
Lab products found in correlation
Morgagni 268d tem, by Thermo Fisher Scientific (1 mentions)
Tecnai g2 spirit transmission em, by Thermo Fisher Scientific (1 mentions)
P35g 1.5 14 cgrd, by MatTek (1 mentions)
Polybed 812 epoxy resin, by Polysciences (1 mentions)
Tecnai transmission electron microscope, by Thermo Fisher Scientific (1 mentions)
Leo 912ab omega electron microscope, by Zeiss (1 mentions)
Cm200 microscope, by Thermo Fisher Scientific (1 mentions)
Emmenu 3, by TVIPS (1 mentions)
5 protocols using sis morada ccd camera
1
Visualizing CpMgm101 Binding to mtDNA
2
Ultrastructural Analysis of Mitochondria in WT and MFN2 KO Hearts
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WT (N = 5) and MFN2 KO hearts (N = 5) were initially extracted and fixed using EM grade 1% paraformaldehyde, 2% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer. Transverse sections of the left ventricle were dissected from fixed hearts, and postfixed in 1% osmium tetroxide and 1.5% potassium ferricyanide for 1 h at 4°C before further staining with aqueous 2% uranyl acetate. Samples were sequentially dehydrated using increasing concentration of ethanol (25%, 50%, 70%, 90%, 100%) and washed with 1,2‐epoxypropane, before embedding in epon resin. For 3D reconstruction, regions of each heart with transverse fibers were sectioned and 40 consecutive 70‐nm sections were collected and further stained with lead citrate. Three representative areas containing intermyofibrillar (IMF) mitochondria were randomly selected in each section series, and imaged across the full 40‐section range (2.8 μm total depth) using a Tecnai G2 Spirit transmission EM (TEM) from FEI, equipped with an Olympus‐SIS Morada CCD camera. Captured images had a pixel size of 2.86 nm in both x and y axes. Video reconstruction was performed on images with the pixel resolution of 2.06 nm in both x and y axes.
3
Correlative Light and Electron Microscopy
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Cells were cultured on gridded coverslip-bottomed dishes (MatTek, P35G-1.5–14-CGRD) to facilitate correlation between light and electron microscopy and processed for the latter as described in [79 ]. Brightfield images of regions of interest were acquired using an Olympus BX50WI upright microscope and TEM images acquired using an FEI tecnai G2 Spirit transmission electron microscope and an Olympus SIS Morada CCD camera.
4
Ultrastructural Analysis of Cultured Cortical Neurons
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Cortical neurons cultured on coverslips were fixed for 30 min at 4°C in 2% glutaraldehyde buffered with 0.1 M sodium phosphate (pH 7.4). Coverslips were rinsed twice in buffer and incubated for 30 min at room temperature in 0.5% osmium tetroxide (OsO4). After rinsing with distilled water, coverslips were stained en bloc with 2% aqueous uranyl acetate for 15 min, dehydrated in ethanol, and embedded in poly/bed 812 epoxy resin (Polysciences) for 24 h. Sections (60 nm thick) were post-stained with uranyl acetate and lead citrate, and viewed with an FEI Tecnai transmission electron microscope at 120 kV of accelerating voltage. Digital images were acquired with an Olympus SIS Morada CCD camera.
5
Cryo-EM and Negative Stain Analysis of EVs
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For Cryo-EM, freshly isolated EVs, PK-treated and non-treated, were plunge frozen as previously described30 (link), using a Vitrobot Mk2 (FEI; Eindhofen, The Netherlands). Images were acquired using the TVIPS EMMENU 3.0 software and a TVIPS TemCam F224 camera on a FEI CM200 microscope operated at 200kV in low dose mode. For negative stain electron microscopy, PK-treated and non-treated vesicles were put on formvar and carbon coated electron microscopy grids (Cu; 200 mesh) for 5 min. Samples were washed (3x using PBS) and then fixed using 2.5% glutaraldehyde in PBS. After two further washes in filtered water, the samples were stained using 2% uranyl acetate for 1.5 min. Images were taken using a SiS Morada CCD-camera (Olympus, Münster, Germany) on a LEO 912AB Omega electron microscope (Carl Zeiss NTS, Jena, Germany) operated at 120kV.
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