C. elegans worms, including E. coli OP50 bacteria, were scraped on to the high-pressure freezing (HPF) specimen carriers. Bacteria served as a filler, minimizing water content and facilitating freezing [46 (link)]. Worms on the carriers were frozen in the high-pressure freezer (Leica EM PACT2). Frozen samples collected on metal carriers were transferred under liquid nitrogen into a pre-frozen cryotube containing 1 ml freeze-substitution solution (2% OsO4 in 100% acetone with 1% lecithin) and finally to a freeze substitution unit (Leica EMAFS) for processing. Samples embedded in Epon EmBed812 resin were ultrasectioned (80nm) with an ultramicrotome (Leica EM UC 6) and placed on copper mesh 300 previously coated with uranyl acetate and lead citrate. Sections were finally examined using a transmission electron microscope (JEOL JEM 2100-Plus 200kV).
Em pact2
Manufactured by Leica camera
Sourced in Germany
The EM PACT2 is a high-performance impact freezer for the rapid freezing of biological samples for transmission electron microscopy. It is designed to freeze specimens at very high cooling rates, preserving their native structure and ultrastructural details. The EM PACT2 utilizes a proprietary freezing process to ensure consistent and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
Afs freeze substitution unit, by Leica camera (4 mentions)
Em afs2, by Leica camera (3 mentions)
Leica afs2, by Leica camera (2 mentions)
Libra 120, by Zeiss (2 mentions)
Tecnai 12, by Thermo Fisher Scientific (2 mentions)
Ultracut uct, by Leica camera (2 mentions)
H 7650, by Hitachi (2 mentions)
Em uc6, by Leica camera (1 mentions)
Em afs, by Leica camera (1 mentions)
Jem 2100 plus 200kv, by JEOL (1 mentions)
Morada g3, by EMSIS (1 mentions)
Ultracut s ultramicrotome, by Leica camera (1 mentions)
Cryotubes, by Sarstedt (1 mentions)
Tecnai f30 microscope, by Thermo Fisher Scientific (1 mentions)
Eagle ccd camera, by Thermo Fisher Scientific (1 mentions)
Axiovert 200, by Zeiss (1 mentions)
Ultrascan 1000 camera, by Ametek (1 mentions)
Fei tecnai g2 20 twin 200 kv lab6 tem, by Thermo Fisher Scientific (1 mentions)
Leica em utc ultramicrotome, by Leica camera (1 mentions)
Formvar coated slot grids, by SPI Supplies (1 mentions)
35 protocols using em pact2
1
High-Pressure Freezing of C. elegans with E. coli OP50
2
Ultrastructural Localization of Antigens
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Animals were anesthetized as above and perfusion fixed in 0.2% glutaraldehyde and 2% paraformaldehyde in 0.1 mol-L–1 PBS (pH 7.4). MA segments (∼2 mm in length) were washed (3 × 5 min) and processed in a Leica EMPACT 2 high-pressure freezer using 0.7% low melting agarose as a cryoprotectant. Samples were then freeze-substituted in a Leica AFS2 into 0.2% uranyl acetate in 95% acetone (from −85 to −50°C) and infiltrated with Lowicryl (at −50°C), before UV polymerization (2 days each at −50 and 20°C; Zechariah et al., 2020 (link)). Conventional transmission electron microscopy (TEM) was conducted using standard procedures (Sandow et al., 2002 (link), 2004 (link)).
Individual serial transverse sections (∼100 nm) were mounted on Formvar-coated slot grids and processed for antigen localization as for confocal immunohistochemistry (per above andTable 2 ). The secondary used was 5 or 10 nmol-L–1 colloidal gold-conjugated antibody (1:40; 2 h) in 0.01% Tween-20. Sections were imaged at x10-40,000 on a JEOL transmission electron microscope at 16 MP (Emsis, Morada G3). Background gold label density was determined from randomly selected (4x) 1 × 1 μm regions per sample of lumen and IEL, compared to the same sized regions of interest in EC profiles.
Individual serial transverse sections (∼100 nm) were mounted on Formvar-coated slot grids and processed for antigen localization as for confocal immunohistochemistry (per above and
3
IPN Ultrastructural Localization
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Isolated IPN from 50 µm vibratome sections were applied to a high pressure freezer (EM PACT2, Leica) in cryoprotectant (30% BSA) and transferred to a freeze substitution unit (EM AFS2, Leica). They were incubated with a substitution medium, containing 1% uranyl acetate, 95% acetone and 5% water, at -85°C, washed in acetone, infiltrated with Lowicryl HM20 and embedded in the resin at -45°C. Ultrathin sections (90 nm) were cut and processed for post-embedding immunolabeling.
4
CLEM protocol for meisosome analysis
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Sample for CLEM experiments were treated as in Johnson et al., 2015 (link). Briefly, the worms were high-pressure frozen (EMPACT2, Leica) and then freeze-substituted (AFS2, Leica) for 20 hr from –130°C to –45°C in an acetone-based cocktail containing 0.2% uranyl acetate, 0.1% tannic acid, and 5% H2O. After 2 hr of acetone washes at –45°C, the samples were infiltrated with gradients of HM20 resin over 9 hr, with pure resin for 18 hr at –45°C and the resin was polymerised under UV for 24 hr at –45°C and for 12 hr at 0°C. Then 350 nm semithin sections were processed as described for TEM tomography above. TEM grids were first analysed at by confocal imaging where a bright-field image is overlaid with the fluorescent image, then analysed in TEM at low magnification. Brightfield, GFP confocal, and TEM images were aligned using Amira software. Several positions with two or three GFP spots were chosen to do a high-magnification tomography, as described above, to reveal the meisosomes.
5
High-Contrast Cryo-EM Imaging of Membranes
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Cells were high-pressure frozen using an EM PACT2 (Leica). Samples were maintained at −90°C in an AFS2 (Leica) freeze substitution device for 44 h in a solution of 20% H2O, 2% osmium tetroxide, and 1% anhydrous glutaraldehyde. A high water content was used as this was found to improve the contrast of RO membranes. The temperature was then raised to 0°C over a period of 22 h through a series of controlled warming phases (identical to those described in reference 21 (link)). Samples were washed with acetone, infiltrated with epoxy resin LX-112 (Ladd Research), and polymerized at 60°C. Sections of 70 nm were collected on EM grids and poststained with uranyl acetate and lead citrate. For tomography, thicker sections of 200 nm were collected. Before poststaining, both sides of the EM grid were incubated with 10-nm colloidal gold beads to serve as fiducial markers during tomogram reconstruction.
6
High-Pressure Freezing and Electron Microscopy
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Larval filets were fixed in 4% PFA, 0.5% glutaraldehyde in 0.1 M PB for 30 min at room temperature. After samples were rinsed in 0.1 M PB, a 1.35‐mm disk was cut out of larval FB and submerged in 20% BSA in DMEM medium with 10% fetal calf serum (FCS). Next tissue disks were loaded in membrane carriers (0.1 mm thick, Leica‐microsystems), frozen with a high‐pressure freezer (Leica EMPACT2), and vitrified at 2,050 bar. The quick freeze substitution protocol was started by transferring the membrane carriers to cryotubes (72.694.005, Sarstedt, Germany) with freeze substitution medium with 0.2% uranyl acetate (#02624‐AB, SPI) in acetone (#1002990500, Merck) and 5% Milli‐Q water at −180°C. Once the cryotubes reached −80°C, the cryotube‐holder was placed on its side and agitated. At −50°C, the carriers were transferred to the pre‐cooled −50°C Leica AFS2 apparatus, washed in 100% acetone, and after a final rinsing in ethanol, infiltrated with lowicryl HM20 (#02628‐AB, SPI), and finally polymerized at −50°C with UV. 200‐nm sections were cut on a Leica Ultracut S ultramicrotome and collected on microscopic slides and immediately followed by 70‐nm sections collected on slot grids (#01805‐F, Ted Pella, USA). The fluorescence image of the last 200‐nm section was overlaid with EM images of the first 70‐nm section using GNU Image Manipulation Program (GIMP).
7
Cryo-Electron Tomography of Yeast Cells
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Ty3-expressing yeast cells were prepared for ET as described by Kukulski et al. (64 (link)). The yeast cell paste was high-pressure-frozen (Empact 2; Leica), processed by freeze substitution, and embedded in Lowicryl resin using an AFS2 (Leica). The samples were sectioned and mounted onto EM grids. Tomographic data were collected using an F30 Tecnai microscope (FEI) equipped with an Eagle CCD camera (FEI) with a pixel size at the specimen level of 11.8 Å. Dual-axis tilt series were collected with a 1° increment in a ±60° range. Tomograms were reconstructed in IMOD (65 (link)).
8
High-Pressure Freezing for Ultrastructural Analysis
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For high-pressure freezing, 4-day-old root tips with indicated treatments were cut and immediately frozen in a high-pressure freezer (EM PACT2, Leica, Germany), followed by subsequent freeze substitution in dry acetone containing 0.1% uranyl acetate at −85 °C in an AFS freeze-substitution unit (Leica, Wetzlar, Germany)60 (link). Infiltration with HM20, embedding, and ultraviolet polymerization were performed stepwise at −10 °C. Immunogold labeling was performed as described previously with anti-VSR antibody at 40 μg/mL, and gold-coupled secondary antibody at 1:50 dilution. TEM examination was done with a Hitachi H-7650 transmission electron microscope with a charge-coupled devise camera operating at 80 kV (Hitachi High-Technologies Corporation, Japan).
9
High Pressure Freezing of Transfected HEK293T Cells
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Transfection efficiency (typically > 50%) and fluorescence level were verified immediately prior to cryofixation using an inverted epifluorescence microscope (Axiovert 200, Zeiss). HEK293T cells were dislodged from the culture plate by gentle tapping and pipetting. The resulting cell suspension (~5 ml) was spun in a benchtop centrifuge at 500 rpm for 1 min. The supernatant was removed and cells were resuspended in 100 μl cryoprotectant (20% BSA in 0.1 M PIPES pH 7.2) pre-warmed to 37°C, then pelleted for 30 s at 10,000 rpm. Alternatively, HEK293T cells were trypsinized and pelleted at 1000 rpm for 2 min in pre-warmed 0.1 M PIPES pH 7.2 containing 20% BSA and 5% fetal bovine serum, which resulted in less clumping of the cells and better resin infiltration. The supernatant was removed and ~1 μl of the resulting cell slurry was loaded into the membrane carrier (Leica), which was then immediately vitrified using a high pressure freezer (EMPACT2, Leica).
10
Ultrastructural Analysis of Symbiotic Protocorms
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Symbiotic protocorms were collected and fixed by high-pressure freezing in a high-pressure freezer (Leica EM PACT2) as described (Li et al. 2018 (link)). The fixed protocorms were exposed to freeze substitution medium (ethanol containing 1% osmium tetroxide, 0.2% glutaraldehyde and 0.1% uranyl acetate) in a Leica Automatic Freeze-Substitution System, then embedded in London Resin White methacrylate resin (London Co., Basingstoke, UK). Ultrathin sections (70–90 nm) were cut by use of the diamond knife of the Leica Reichert Ultracut S system (Leica Microsystems GmbH1) and placed on formvar-coated nickel grids for observation under a Philip CM 100 transmission electron microscope (FEI Company2) at 80 kV.
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