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Em cpc

Manufactured by Leica
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Sourced in Germany, Japan

The EM CPC is a critical point dryer for electron microscopy sample preparation. It allows the removal of liquid from specimens while preserving their structure.

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Lab products found in correlation

Jem 3100fef, by JEOL (4 mentions) Fr 7000a, by Hitachi (3 mentions) Lowicryl hm20 resin, by Electron Microscopy Sciences (3 mentions) Em afs, by Leica (3 mentions) Afs freeze substitution instrument, by Leica (2 mentions) Hdt 400, by JEOL (2 mentions) Model 626 dh, by Ametek (2 mentions) H 7650, by Hitachi (2 mentions) H 7650 transmission electron microscope, by Hitachi (2 mentions) Vt1000, by Leica (2 mentions) Falcon 2, by Thermo Fisher Scientific (1 mentions) Titan krios electron microscope, by Thermo Fisher Scientific (1 mentions) Jem 1010, by JEOL (1 mentions) Ni nta nanogold beads, by Nanoprobes (1 mentions) Ultrascan 2000, by Ametek (1 mentions) Model 794, by Ametek (1 mentions) Eclipse 50i fluorescent microscope, by Nikon (1 mentions) Uranyl acetate, by Polysciences (1 mentions) Lowicryl hm20 resin, by Polysciences (1 mentions) Sucrose, by Merck Group (1 mentions)

29 protocols using em cpc

1

Cryogenic X-ray Tomography of Migrating Cells

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CEM cells were seeded on gold quantifoil R 2/2 holey film microscopy grids (Au-G200F1) coated with poly-L-lysine and allowed to migrate for 30 min in RPMI medium containing 40 ng/mL SDF-1α. Samples were vitrified by plunge freezing in a Leica EM CPC. Vitrified grids were transferred to Mistral (ALBA light source) [32 (link), 33 (link)] beamline in ALBA synchrotron. For image acquisition, we used X rays with 520 eV photon energy, and X-ray projections taken at liquid nitrogen temperature with 1° tilt steps.
The zone plate objective has an outermost zone width of 40 nm; tilt series alignment reconstruction and segmentation were performed as described in [34 (link)]. We used USFC Chimera software to join the three tomograms (Supporting Information Fig. 2) reconstruction generating a whole cell final tomogram.
Ramírez-Santiago G., Robles-Valero J., Morlino G., Cruz-Adalia A., Pérez-Martínez M., Zaldivar A., Torres-Torresanó M., Chichón F.J., Sorrentino A., Pereiro E., Carrascosa J.L., Megías D., Sorzano C.O., Sanchez-Madrid F, & Veiga E. (2016). Clathrin regulates lymphocyte migration by driving actin accumulation at the cellular leading edge. European journal of immunology, 46(10), 2376-2387.
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2

Double-Immunogold Labeling of Rat Brain

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Animals used for postembedding, double-immunogold localization were prepared as described previously (Petralia and Wang, 2021 (link)). Briefly, two male, adult Sprague Dawley rats were perfused with phosphate buffer, followed by perfusion with 4% paraformaldehyde + 0.5% glutaraldehyde in phosphate buffer, and then the brains were vibratomed, cryoprotected in glycerol overnight, frozen in a Leica EM CPC (Vienna, Austria), and embedded in Lowicryl HM-20 resin in a Leica AFS freeze-substitution instrument. Thin sections were incubated in 0.1% sodium borohydride + 50 mM glycine in Tris-buffered saline plus 0.1% Triton X-100 (TBST), then in 10% normal goat serum (NGS) in TBST, and then with 2 primary antibodies together in 1% NGS/TBST (overnight); then they were incubated with the 2 immunogold-conjugated secondary antibodies (5+15 nm; Ted Pella, Redding, CA, USA) in 1% NGS in TBST with 0.5% polyethylene glycol (20,000 MW) and stained with uranyl acetate and lead citrate. Controls on sections from the same two rats, labeled with the 2 secondary antibodies but without the primary antibodies, showed only rare gold and no colocalization.
Murphy J.G., Gutzmann J.J., Lin L., Hu J., Petralia R.S., Wang Y.X, & Hoffman D.A. (2022). R-type voltage-gated Ca2+ channels mediate A-type K+ current regulation of synaptic input in hippocampal dendrites. Cell reports, 38(3), 110264.
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3

Cryo-EM Imaging of RnQV1-W1118 Particles

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Purified RnQV1-W1118 empty particles (5 μl) were applied to R2/2 300 mesh copper-rhodium grids (Quantifoil Micro Tools, Germany) and vitrified using a Leica EM CPC cryofixation unit. Data were collected on a FEI Titan Krios electron microscope operating at 300 kV and images recorded on a FEI Falcon II detector at a calibrated magnification of 104,478, yielding a pixel size of 1.34 Å. A dose rate of 28 electrons/Å2/s and 1.4 s exposure time were used to record 1,125 24-frame movies with a defocus range of 0.7 to 3.5 μm.
Mata C.P., Luque D., Gómez-Blanco J., Rodríguez J.M., González J.M., Suzuki N., Ghabrial S.A., Carrascosa J.L., Trus B.L, & Castón J.R. (2017). Acquisition of functions on the outer capsid surface during evolution of double-stranded RNA fungal viruses. PLoS Pathogens, 13(12), e1006755.
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4

Exosomal Characterization via TEM Imaging

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The exosomal fraction isolated from pFF was resuspended in NaHCa buffer (30 mM HEPES, 100 mM NaCl, 2 mM CaCl2, pH 7.4). The resuspended exosomal
fraction was applied on a 200-mesh copper microgrid covered with a formvar support film, which had been pre-treated with soft plasma etching equipment (SEDE-AF,
Meiwafosis, Tokyo, Japan) to obtain a hydrophilic surface. The microgrid was electron-stained with 1% uranium acetate solution for 10 min and then with lead
acetate solution for 10 min. Finally, the microgrid was observed with a transmission electron microscope (JEM-1010; JEOL, Tokyo, Japan).
For cryogenic TEM (Cryo-TEM), the resuspended exosomal fraction in NaHCa buffer was applied on a 200-mesh copper microgrid that had been pre-treated with a
glow-discharger (HDT-400, JEOL) to obtain a hydrophilic surface. The microgrid was immediately plunged into liquid propane using a specimen preparation machine
(EM CPC; Leica) and then transferred to a cryo-transfer holder (Model 626-DH; Gatan, Pleasanton, CA, USA). Finally, the microgrid was observed with a
transmission electron microscope (JEM-3100FEF; JEOL) at an acceleration voltage of 300 kV in zero-loss imaging mode. The temperature of the microgrid was
maintained below –140°C during the observation.
MATSUNO Y., ONUMA A., FUJIOKA Y.A., YASUHARA K., FUJII W., NAITO K, & SUGIURA K. (2017). Effects of exosome-like vesicles on cumulus expansion in pigs in vitro. The Journal of Reproduction and Development, 63(1), 51-58.
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5

Btub Filament Visualization with Bklc

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The images obtained for Btub filaments in absence or presence of Bklc were obtained by 2 consecutive cycles of polymerization in order to remove protein noise that could be generated from non-polymerization competent molecules. The Ni-NTA-nanogold beads (5 nm; purchased from Nanoprobes) were added after the second polymerization and used at a final concentration of 50 nM.
For the SUV images with or without Bklc, experiments were carried out at room temperature, and the SUVs were incubated with or without Bklc for 45–60 minutes using concentrations of 2.5 mM and 4 M for SUVs and Bklc respectively in 20 mM TrisHCl pH 7.5 and 150 mM NaCl. The Ni-NTA-nanogold beads (5 nm; purchased from Nanoprobes) were added just prior to sample freezing and used at a final concentration of 50 nM.
For plunge freezing of samples, a 5 μl drop of sample was deposited onto 300 mesh holey carbon copper grids (Ted Pella). The excess of solution was manually blotted using a Whatman filter paper and the grid was plunge-frozen in liquid ethane using a Leica EM-CPC.
Akendengue L., Trépout S., Graña M., Voegele A., Janke C., Raynal B., Chenal A., Marco S, & Wehenkel A.M. (2017). Bacterial kinesin light chain (Bklc) links the Btub cytoskeleton to membranes. Scientific Reports, 7, 45668.
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6

Double-Immunogold Labeling of Rat Brain

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Animals used for postembedding, double-immunogold localization were prepared as described previously (Petralia and Wang, 2021 (link)). Briefly, two male, adult Sprague Dawley rats were perfused with phosphate buffer, followed by perfusion with 4% paraformaldehyde + 0.5% glutaraldehyde in phosphate buffer, and then the brains were vibratomed, cryoprotected in glycerol overnight, frozen in a Leica EM CPC (Vienna, Austria), and embedded in Lowicryl HM-20 resin in a Leica AFS freeze-substitution instrument. Thin sections were incubated in 0.1% sodium borohydride + 50 mM glycine in Tris-buffered saline plus 0.1% Triton X-100 (TBST), then in 10% normal goat serum (NGS) in TBST, and then with 2 primary antibodies together in 1% NGS/TBST (overnight); then they were incubated with the 2 immunogold-conjugated secondary antibodies (5+15 nm; Ted Pella, Redding, CA, USA) in 1% NGS in TBST with 0.5% polyethylene glycol (20,000 MW) and stained with uranyl acetate and lead citrate. Controls on sections from the same two rats, labeled with the 2 secondary antibodies but without the primary antibodies, showed only rare gold and no colocalization.
Wood G.E., Dutro S.M, & Totten P.A. (1999). Target Cell Range of Haemophilus ducreyi Hemolysin and Its Involvement in Invasion of Human Epithelial Cells. Infection and Immunity, 67(8), 3740-3749.
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7

Cryo-EM Imaging of Viral Proteins

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NTR and TR-TLP samples (5 µl) were applied to one side of acetone-washed Quantifoil R 2/2 holey grids, blotted, and plunged into liquid ethane using a Leica EM CPC cryo-fixation unit. Cryo-EM images were recorded in low-dose conditions (∼10 e−/Å2), in a Tecnai G2 electron microscope operating at 200 kV. For SA11 NTR and TR samples, micrographs were recorded at a nominal magnification of 50,000X. SA11 NTR-TLP produced in the absence of leupeptin and OSU samples were imaged on a FEI Eagle 4k CCD at a detector magnification of 67,873X (2.21 Å/pixel sampling rate).
Rodríguez J.M., Chichón F.J., Martín-Forero E., González-Camacho F., Carrascosa J.L., Castón J.R, & Luque D. (2014). New Insights into Rotavirus Entry Machinery: Stabilization of Rotavirus Spike Conformation Is Independent of Trypsin Cleavage. PLoS Pathogens, 10(5), e1004157.
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8

Characterization of Extracellular Vesicles by NTA and Cryo-TEM

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Size distribution and an estimated concentration of isolated EVs were determined using nanoparticle tracking analysis (NTA; NanoSight NS300, Malvern Instruments Limited, Worcestershire, UK) following the manufacturer’s instructions. EV samples were diluted at 1:40 with sterile and filtered PBS, and three videos of 60 s at 25.0 frames per second (FPS) at a camera level of 10 were recorded. The mean values for size (nm) and concentration (particles/mL) of the three replicates were considered as the final measurement.
Additionally, EV-enriched fractions were examined using cryo-transmission electron microscopy (cryo-TEM), as previously described [32 (link)]. Briefly, vitrified specimens were prepared by placing 3 μL of a sample on a Quantifoil. 1.2/1.3 TEM grid, blotted to a thin film, and plunged into liquidethane-N2(l) in the Leica EM CPC cryo-work station. The grids were analyzed with a Jeol JEM 2011 transmission electron microscope, and images were recorded on a Gatan Ultrascan 2000 cooled charge-coupled device (CCD) camera with the Digital Micrograph software package (version 3.x Ametek, Berwyn, PA, USA).
Solaguren-Beascoa M., Gámez-Valero A., Escaramís G., Herrero-Lorenzo M., Ortiz A.M., Minguet C., Gonzalo R., Bravo M.I., Costa M, & Martí E. (2023). Phospho-RNA-Seq Highlights Specific Small RNA Profiles in Plasma Extracellular Vesicles. International Journal of Molecular Sciences, 24(14), 11653.
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9

Freeze-Replica TEM Analysis of Photoresist

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FF-TEM analysis
was performed using
a Hitachi High-Tech H-7650 instrument to observe the photoresist dispersion
state in bulk. The samples were rapidly frozen in liquid propane (<−170
°C) using a quick freezer (EM CPC, Leica) and cut with a glass
knife. The replica was prepared by exposing the cross-section of the
frozen dispersion to platinum vapor, followed by treatment with carbon
vapor to build the replica using a freeze-replica apparatus (FR-7000A,
Hitachi High-Tech). The replica was washed with a chloroform/methanol
(v/v = 2/1) solution, acetone, and water. The replica was visualized
at an acceleration voltage of 100 kV.
Hanzawa M., Ogura T., Tsuchiya K., Akamatsu M., Sakai K, & Sakai H. (2023). Anti-adsorption Mechanism of Photoresist by Pluronic Surfactants: An Insight into Their Adsorbed Structure. Langmuir, 39(22), 7876-7883.
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10

Cryo-TEM Imaging of Polymer Solution

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The specimen for cryo-TEM was prepared by rapid freezing of a polymer solution at a concentration of 10 mg/mL. A 200 mesh copper microgrid was used and pretreated with a glow-discharger (HDT-400, JEOL, Tokyo, Japan) to make the microgrid surface hydrophilic. An aliquot (3.0 µL) of a polymer sample was placed on the mesh and immediately plunged into liquid propane using a specimen preparation machine (EM CPC, Leica, Wetzlar, Germany). The temperature of the specimen was maintained below −140 °C during the observation using a cryo-transfer holder (Model 626.DH, Gatan, Pleasanton, CA, USA). Microscopic observations were carried out using a transmission electron microscope (JEM-3100FEF, JEOL, Tokyo, Japan) at an acceleration voltage of 300 kV in zero-loss imaging mode. The microscopic image was recorded using a CCD camera (Model 794, Gatan, Pleasanton, CA, USA) installed in the microscope.
Oda Y., Yasuhara K., Kanaoka S., Sato T., Aoshima S, & Kuroda K. (2018). Aggregation of Cationic Amphiphilic Block and Random Copoly(vinyl ether)s with Antimicrobial Activity. Polymers, 10(1), 93.
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