Immunoelectron microscopy was performed as described in a previous study45 (link) with some modifications. Arabidopsis root tips were frozen in a high-pressure freezing machine (EM PACT; Leica Microsystems, Wetzlar, Germany). The samples were freeze-substituted with 0.25% glutaraldehyde and 0.1% uranyl acetate in 100% acetone at −80 °C for 4 days, and then gradually warmed (EM AFS; Leica Microsystems). The samples were washed with 100% acetone, infiltrated with methanol, and then embedded in LR White resin (London Resin Company Ltd., London, UK). Ultrathin sections (70–80 nm) on formvar-coated nickel grids were labeled with anti-GFP antibody (1:50, A11122; Thermo Fisher Scientific, Waltham, MA, USA) in 50 mM Tris-buffered saline (TBS). After washing with TBS, sections were labeled with 12-nm colloidal gold particles coupled to goat anti-rabbit IgG (1:20, AB_2338016; Jackson ImmunoResearch, West Grove, PA, USA). The sections were stained with 4% uranyl acetate for 20 min and then examined under a transmission electron microscope (JEM-1400; JEOL, Tokyo, Japan) at 80 kV.
Em pact
Manufactured by Leica
Sourced in Germany
The EM-Pact is a high-precision freezing device used for preparing biological samples for electron microscopy. It rapidly freezes samples at extremely low temperatures, preserving their native structure and composition. The EM-Pact is designed to ensure consistent, reliable results in sample preparation for advanced microscopy techniques.
Automatically generated - may contain errors
Lab products found in correlation
Em afs, by Leica (3 mentions)
Tecnai spirit, by Thermo Fisher Scientific (3 mentions)
A11122, by Thermo Fisher Scientific (1 mentions)
Jem 1400, by JEOL (1 mentions)
Goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Anti gfp antibody, by Thermo Fisher Scientific (1 mentions)
Uranyl acetate, by Polysciences (1 mentions)
Diamond knife, by Electron Microscopy Sciences (1 mentions)
Megaview g2 ccd camera, by Olympus (1 mentions)
Vibratome 1000, by Leica (1 mentions)
Tem 910 transmission electron microscope, by Zeiss (1 mentions)
11 protocols using em pact
1
Immunoelectron Microscopy of Arabidopsis
2
High-Pressure Freezing of Yeast Cells
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Fresh baker's yeast cells (Saccharomyces cerevisiae) are centrifuged into a soft pellet after being cultured in a standard culture medium and subsequently mixed with 20% Dextran (40 kDa; Sigma # 31389) as filler. The mixture of yeast cells and Dextran is pushed and pulled through Leica EM PACT copper tubes several times before freezing in the EM PACT (Leica Microsystems, Vienna, Austria), to ensure complete filling without air spaces. Preparation of yeast cells in planchets follows the method described in detail in De Winter et al. (2013 ). As planchets, 100‐µm‐deep Leica membrane carriers are used. High‐pressure freezing is also carried out in an EM PACT unit.
3
Subcellular Localization of GLB-12 in C. elegans
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To analyse the subcellular localization of GLB-12, the GLB-12 translational reporter in combination with anti-GFP antibodies was used. N2 WT worms were included as negative control.
Copper membrane carriers (1.5 mm × 0.2 mm) (Leica, Microsystems, Vienna, Austria) treated with 1% lecithin were used and filled with 20% (w/v) Bovine Serum Albumin (BSA). Three to four one-day-old adult nematodes were transferred to the membrane carrier, immersed in the BSA and then immediately frozen in a high-pressure freezer (EM PACT, Leica, Microsystems).
Freeze substitution was carried out using a Leica EM AFS (Leica, Microsystems). Carriers were transferred from EM PACT to AFS under liquid nitrogen and placed in an eppendorf filled with dry acetone. Over a period of 5 days, animals were freeze-substituted as follows: −90 °C for 27 h, 2 °C per hour increase for 15 h, −60 °C for 12 h, 2 °C per hour increase for 15 h, −30 °C for 32 h, 2 °C per hour increase for 17 h. At 4 °C carriers were rinsed 3 times with dry acetone for 20 min each time.
Copper membrane carriers (1.5 mm × 0.2 mm) (Leica, Microsystems, Vienna, Austria) treated with 1% lecithin were used and filled with 20% (w/v) Bovine Serum Albumin (BSA). Three to four one-day-old adult nematodes were transferred to the membrane carrier, immersed in the BSA and then immediately frozen in a high-pressure freezer (EM PACT, Leica, Microsystems).
Freeze substitution was carried out using a Leica EM AFS (Leica, Microsystems). Carriers were transferred from EM PACT to AFS under liquid nitrogen and placed in an eppendorf filled with dry acetone. Over a period of 5 days, animals were freeze-substituted as follows: −90 °C for 27 h, 2 °C per hour increase for 15 h, −60 °C for 12 h, 2 °C per hour increase for 15 h, −30 °C for 32 h, 2 °C per hour increase for 17 h. At 4 °C carriers were rinsed 3 times with dry acetone for 20 min each time.
4
Cryo-EM Sample Preparation for Yeast Cells
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Cultures were grown in 200 ml of yeast extract/peptone/dextrose (YPD) to an OD600 of ∼0.8. Samples were frozen on the EM-Pact (Leica) at ∼2050 bar, transferred under liquid nitrogen into 2% osmium tetroxide/0.1% uranyl acetate/acetone/2% water, and then transferred to the Leica AFS2. The freeze-substitution protocol started samples at −90°C for 72 h, up 5°C/h, −20°C for 12 h, up 5°C/h, to 0°C for 5 h. Samples were then removed from the AFS2 and allowed to come to room temperature for 1 h before going through three changes of acetone over 1 h. They were removed from the planchettes and stepwise embedded in acetone/Epon/Araldite mixtures to the final 100% Epon/Araldite over several days. Sections were then cut at 80 nm on a Leica UC6, stained with uranyl acetate and Sato's lead, and imaged on a Tecnai Spirit (FEI).
5
Freeze Substitution of Yeast Cells
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Cells were grown overnight at 23°C in YPD, then shifted to 37°C for 4 h. Cells were quickly harvested and frozen on the Leica EM-Pact (Wetzlar, Germany) at ∼2050 bar, transferred under liquid nitrogen into 2% osmium tetroxide/0.1% uranyl acetate/acetone, and transferred to the Leica AFS. The freeze substitution protocol was as follows: −90°C for 16 h, raised 4°C/h for 7 h, −60°C for 19 h, raised 4°C/h for 10 h, and −20°C for 20 h. Samples were then removed from the AFS, placed in the refrigerator for 4 h, and then allowed to incubate at room temperature for 1 h. Samples went through three changes of acetone over 1 h and were removed from the planchettes. They were embedded in acetone/Epon mixtures to final 100% Epon over several days in a stepwise procedure as previously described (McDonald, 1999 ). Sixty-nanometer serial thin sections were cut on a Leica UC6, stained with uranyl acetate and Sato’s lead, and imaged on a FEI Technai Spirit (Hillsboro, OR).
6
High-pressure Freezing and Electron Microscopy
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Yeast cell culture grown in SMDGE I- medium for 24 h at 30 °C was processed as described previously (57 (link)). Briefly: cells were concentrated by suction filtration, loaded in a flat specimen carrier, and quickly frozen in Leica EM PACT high-pressure freezer. Frozen samples were freeze substituted in acetone supplemented with 3% glutaraldehyde (EMS; 10% stock in acetone), 0.1% uranyl acetate (Polysciences; 20% methanolic stock), 1% OsO4 (EMS; 10% stock in acetone), and 1% water in Leica AFS machine and then embedded in Lowicryl HM20 resin (EMS). Ultrathin sections (70 nm) were cut with Ultracut S ultramicrotome equipped with a diamond knife (45◦; Diatome) and placed on copper formvar-coated grids. Sections were examined in an FEI Morgagni 268(D) transmission electron microscope at 80 kV. Images were captured with Mega View G2 CCD camera (Olympus).
7
High-pressure Freezing and Electron Microscopy
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ndc1-A290E cells were grown overnight at 23°C and then shifted into a pre-warmed 37°C water bath for 4 h. Cells were quickly harvested and frozen on a high-pressure freezer (EM-Pact; Leica) at ∼2050 bar, transferred under liquid nitrogen into 2% osmium tetroxide/0.1% uranyl acetate/acetone, and transferred to an automatic freeze substitution (AFS) chamber (Leica). The freeze substitution protocol was as follows: −90° for 16 h, raised 4°/h for 7 h; −60° for 19 h, raised 4°/h for 10 h; and −20° for 20 h. Samples were then removed from the AFS, placed in the refrigerator for 4 h, and then allowed to incubate at room temperature for 1 h. Samples went through three changes of acetone over 1 h and were removed from the planchettes. They were embedded in acetone/Epon mixtures to final 100% Epon over several days in a stepwise procedure as described previously (McDonald, 1999 (link)). 60-µm 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).
8
Structural Analysis of Mouse Heart Models
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Mice were maintained under Home Office regulations, Project Licence 70/06495 in accordance with the Animal Scientific Procedures Act, UK, 1986.
Sections from mouse heart were prepared as previously described (Wilson et al. 2014 (link)). The types of specimen used were the mouse model of DCM, the MLP KO, and its control strain, SV129. For some of the tomography, specimens were high-pressure frozen. Hearts taken from MLP KO and control mice were fixed in 4 % PFA in PBS and washed in PBS as standard procedure. 100 μm sections were cut on a Leica Vibratome 1000S and then small pieces were high pressure frozen in a Leica EM PACT. Frozen samples were freeze substituted in 2 % osmium in acetone, washed in acetone and embedded in Araldite (Bennett et al. 2002 (link)).
Sections from mouse heart were prepared as previously described (Wilson et al. 2014 (link)). The types of specimen used were the mouse model of DCM, the MLP KO, and its control strain, SV129. For some of the tomography, specimens were high-pressure frozen. Hearts taken from MLP KO and control mice were fixed in 4 % PFA in PBS and washed in PBS as standard procedure. 100 μm sections were cut on a Leica Vibratome 1000S and then small pieces were high pressure frozen in a Leica EM PACT. Frozen samples were freeze substituted in 2 % osmium in acetone, washed in acetone and embedded in Araldite (Bennett et al. 2002 (link)).
9
Cryoimmobilization and Ultrastructural Analysis
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Explants from Nicotiana leaves or Arabidopsis seedlings were excised under a stereomicroscope and transferred to aluminium planchette with a 200 μm-deep cavity that was subsequently filled with yeast (Saccharomyces cerevisiae) paste and cryoimmobilized immediately with a high pressure freezer (EM Pact (Leica)). Freeze substitution of frozen samples was performed in an automatic freeze substitution system (EM AFS (Leica)) with acetone containing 2% (w/v) osmium tetroxide and 0.1% (w/v) uranyl acetate, for 3 d at −90 °C. On the fourth day, the temperature was raised by 5 °C per hour to room temperature. At this temperature, samples were rinsed in acetone, infiltrated with Epon (Nicotiana samples, Figure 4 ) or Spurr (Arabidopsis samples, Figure 5 ) resin for 2 d, embedded in a thin layer of the same resin and polymerized at 60 °C for 48 h.
10
Ultrastructural Analysis of Drosophila Embryos
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For conventional transmission electron microscopy (TEM), previously fixed embryos were post fixed in 1% osmium tetroxide in 0.1M sodium cacodylate, embedded in Epon resin, and ultrathin 50–70 nm sections examined (80 kV; Tecnai Spirit; FEI).
For HPF-FS electron microscopy, stage 14 embryos were frozen on a Leica EM-Pact at 2100 bar, and transferred under liquid nitrogen into 2% osmium tetroxide/0.1% uranyl acetate/acetone to an automated freeze-substitution apparatus (AFS) (Leica, Vancouver). Samples were embedded in an acetone/Epon mixture and transferred to 100% Epon as described [38] (link). Sectioning and staining were done as described for conventional EM, and serial section images aligned using AutoAligner (Bitplane).
For HPF-FS electron microscopy, stage 14 embryos were frozen on a Leica EM-Pact at 2100 bar, and transferred under liquid nitrogen into 2% osmium tetroxide/0.1% uranyl acetate/acetone to an automated freeze-substitution apparatus (AFS) (Leica, Vancouver). Samples were embedded in an acetone/Epon mixture and transferred to 100% Epon as described [38] (link). Sectioning and staining were done as described for conventional EM, and serial section images aligned using AutoAligner (Bitplane).
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