Acute brain slices were sampled by collecting 2 mm diameter cortical tissue biopsies. These were incubated in cryoprotectant (20% w/v 40,000 Dextran22 (link) in NMDG-HEPES solution) for ~45 min at RT. 100 μm deep wells of the specimen carrier type A (Leica, 16770152) were filled with cryoprotectant, tissue biopsies were carefully placed inside and covered with the flat side of the lipid-coated specimen carrier type B (Leica, 16770153) and high-pressure frozen (~2000 bar, −188 °C) using a Leica EM ICE.
Em ice
Manufactured by Leica camera
Sourced in France
The EM ICE is a cryogenic transmission electron microscope (cryo-TEM) designed for high-resolution imaging of biological samples. It provides the capability to visualize and analyze the structure of macromolecules, cells, and other biological specimens in their native, hydrated state.
Automatically generated - may contain errors
Lab products found in correlation
Em vct500, by Leica camera (5 mentions)
Em ace900, by Leica camera (5 mentions)
Em vcm, by Leica camera (5 mentions)
Em afs2, by Leica camera (3 mentions)
Geminisem, by Zeiss (3 mentions)
Type a 6 mm cu au carrier, by Leica camera (2 mentions)
Tecnai spirit, by Thermo Fisher Scientific (1 mentions)
Afs2 chamber, by Leica camera (1 mentions)
Talos l120c tem, by Thermo Fisher Scientific (1 mentions)
Em uc7 ultramicrotome, by Leica camera (1 mentions)
Quetol 812 epoxy resin, by Nisshin EM (1 mentions)
Ultra plus fesem, by Zeiss (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Ultra 55 sem, by Zeiss (1 mentions)
Ht7800 tem, by Hitachi (1 mentions)
Em uc7, by Leica camera (1 mentions)
Hm20 resin, by Polysciences (1 mentions)
Afs2 unit, by Leica camera (1 mentions)
Tecnai 12 tem, by Ametek (1 mentions)
Oneview digital camera, by Ametek (1 mentions)
22 protocols using em ice
1
High-Pressure Freezing of Brain Slices
2
Freeze Substitution Protocol for TEM
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Cells were grown to logarithmic phase in YPGR, washed with YPD, and grown in YPD for 12 h. Cells were harvested and frozen on a high-pressure freezer (EM ICE; Leica) at ∼2,050 bar, transferred under liquid nitrogen into 2% osmium tetroxide/0.2% uranyl acetate/acetone, and transferred to an automatic freeze substitution (AFS2) chamber (Leica) at −140°C. The freeze substitution protocol was as follows: −90° raised to −80° over 60 h, −80° raised to −60 over 5 h, held at −60 for 4 h, −60 to −20° over 5 h, held at −20° for 4 h, −20° to 0° over 4 h, and held at 0° for 5 h. Next, samples were removed from the AFS2, incubated at room temperature for 1 h, washed with acetone four times over 1 h, and removed from the planchettes. Samples were embedded in acetone/epon mixtures to final 100% epon as described previously (McDonnald, 1999 ). Finally, 50-nm serial thin sections were cut on an ultramicrotome (model UC6; Leica), stained with uranyl acetate and Sato’s lead, and imaged on a transmission electron microscope (Tecnai Spirit; FEI).
3
Mitochondrial Morphology Analysis in Hermaphroditic Germline
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The mitochondrial morphology of the hermaphroditic germline, oocyte and embryo was analyzed with TEM. Samples were prepared following a high-pressure freezing standard protocol. Briefly, worms were picked into 200 μL M9 buffer with 20% BSA and frozen with a high-pressure freezing machine (Leica EM ICE). Then, the samples were freeze-substituted in a Leica EM AFS2 freeze-substitution unit with medium (containing 1% osmium tetroxide, 0.1% uranyl acetate, 10% methanol in acetone) using a program as previously described.62 (link) At the end of freeze-substitution, the temperature was increased to 20 °C. The samples were washed in pure acetone 3 times before being infiltrated with increasing concentrations of Epon resin (diluted with acetone): 25% Epon resin for 30 min, 33% for 210 min, 50% for 12 h, 75% for 240 min, and pure resin four times in a 24-h period. The samples were embedded in a mold and cured for 2 days at 60 °C.
For TEM imaging, samples were cut into 70-nm-thick sections using a Leica EM UC7 ultramicrotome and collected on copper grids. The sections were counterstained with 2% uranyl acetate and Sato’s triple lead. All imaging was carried out on a Thermo Fisher Scientific Talos L120C TEM at 80 kV equipped with a 2k × 2k Ceta CCD camera. The percentages of donut-shaped mitochondria and non-donut-shaped mitochondria were quantified and analyzed in a blind manner.
For TEM imaging, samples were cut into 70-nm-thick sections using a Leica EM UC7 ultramicrotome and collected on copper grids. The sections were counterstained with 2% uranyl acetate and Sato’s triple lead. All imaging was carried out on a Thermo Fisher Scientific Talos L120C TEM at 80 kV equipped with a 2k × 2k Ceta CCD camera. The percentages of donut-shaped mitochondria and non-donut-shaped mitochondria were quantified and analyzed in a blind manner.
4
Kidney Tissue Fixation and Embedding
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At the end of the feeding, kidney tissues of mice fed with an HPD or normal diet were obtained following euthanasia, cut into small pieces in Krebs–Ringer HEPES buffer, and fixed with either immersion fixation or HPF using EM ICE (Leica). In the immersion fixation, some pieces of kidney tissues were immersed in buffered formalin, embedded in paraffin wax, and observed using von Kossa staining after sectioning, as previously described20 (link). The frozen tissues were embedded in epoxy resin as previously described4 (link). Briefly, the tissues were freeze-substituted in acetone containing 2% osmium tetroxide (Nissin EM) using EM AFM2 (Leica). The substitution medium was replaced with pure acetone after increasing the temperature and incubating at room temperature, and the samples were embedded in Quetol 812 epoxy resin (Nisshin EM Co.).
5
Correlative Microscopy of Lecithinase
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WT BMDMs left untreated or treated with AF568‐lecithinase for 90 min were washed with PBS. Cells were pelleted and high pressure frozen (Leica EM‐ICE). Cells were then freeze substituted and embedded in resin (Leica EM AFS2). The cells were ultra‐microtomed at 300 nm thin sections (Leica EM UC7) and then viewed on confocal microscope (ZEISS LSM800) to capture the fluorescence signal within the cells and on scanning electron microscope (Zeiss UltraPlus FE SEM) to capture ultrastructure. The SEM images were captured at 2 kV accelerating voltage using the energy selective backscattered (EsB) detector. After the SEM images were captured, the correlation of AF568‐lecithinase into subcellular structures was then performed via a shuttle‐and‐find system (ZEISS).
6
High-Pressure Freezing and Freeze Substitution of Arabidopsis Root Tips
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High-pressure freezing and freeze substitution (HPFS) were performed as described previously (63 (link)). Briefly, 5-d-old Arabidopsis root tips were frozen in a high-pressure freezer (Leica EM ICE). High-pressure frozen root tips were transferred to an AFS (Leica Microsystems) and freeze-substituted in dry acetone containing 0.1% uranyl acetate at −85 °C for 48 h. Infiltration with HM20, embedding, and ultraviolet polymerization were performed stepwise at −45 °C. Immunogold labeling was performed as described previously (64 (link)). GFP antibodies were diluted at 40 μg/mL in phosphate-buffered saline (PBS) with 1% bovine serum albumin (BSA), and gold-coupled rabbit secondary antibodies were at 1:40 dilution in PBS with 1% BSA.
7
High-Pressure Freezing for Cryoprotection
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Following incubation with 15NH4+, animals were carefully detached and immediately transferred to type A 3 mm Au-coated Cu-carriers (large cavity, 200 μm deep, Wohlwend) filled with dextran 40 in M-solution (20% w/v), which serves as cryoprotectant and filler. Each carrier containing one animal was immediately covered with the flat side of a type B Al-carrier previously coated in 1-hexadecene. The sample was immediately high pressure frozen using an EM ICE (Leica) device, which creates an optimum pressure of 2100 bar on the sample within a few milliseconds followed by immediate rapid cooling [45 (link)]. The hexadecene coating helps to detach the type B carrier from the type A carrier after high pressure freezing without damaging the surface of the vitrified sample that remains in the type A Cu-carrier during all further steps.
8
Cryoimmobilization and Cryo-SEM Imaging
9
Ultrastructural Analysis of Adult Animal Tissues
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Adult animals were fixed using a high-pressure freezer (EM ICE, Leica). Freeze-substitution was carried out in anhydrous acetone containing 1% osmium tetroxide and 0.1% uranyl acetate dihydrate. The samples were sequentially placed at −90 °C for 72 h, −60 °C for 10 h and −30 °C for 10 h, and 0 °C for 5 h in a freeze-substitution unit EM AFS2 (Leica, Germany). After 3 washes (20 min each) with fresh cold anhydrous acetone, the samples were infiltrated with Embed-812 resin. Samples were embedded at 60 °C for 48 h and cut into 70 nm sections with a microtome (EM UC7, Leica, Germany). After electron staining with uranyl acetate and lead citrate, sections were observed with a HT7800 TEM (HITACHI, Japan) operating at 80 kV.
10
High-Pressure Freezing and Electron Microscopy
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Exponentially growing cells were pelleted, loaded into 3 mm, 100 µm, or 200 µm carriers and high-pressure frozen using a Leica EM ICE. Samples were freeze substituted in a Leica AFS2 unit in 1% uranyl acetate +1% water in acetone. Samples were then maintained at − 45 °C for the remainder of the processing. Samples were washed with acetone and then ethanol for 15 min each, then infiltrated with HM20 resin (PolySciences) according to the following schedule: 25% resin in ethanol for 4 h, 50% resin in ethanol overnight, 75% resin in ethanol for 5 h, 100% resin for 6 h, 100% resin overnight, 100% resin for 8 h. Samples were then polymerized with UV light for 48 h, and gradually warmed to 0 °C after the first 24 h of polymerization. 90 nm ultranthin sections were acquired using a Diatome diamond knife with a Leica UC7 ultramicrotome and transferred to formvar-coated 100-mesh copper grids. Sections were post-stained for 10 min with 2% uranyl acetate, washed by passing over a series of warm water droplets, and then stained with Reynold’s lead citrate, washed, and dried. Grids were imaged in a FEI Tecnai 12 TEM operated at 120 kV using a Gatan OneView digital camera.
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