Hpm 010 high pressure freezing machine

Manufactured by Leica

Sourced in Switzerland

The HPM 010 is a high-pressure freezing machine manufactured by Leica. It is designed to rapidly freeze samples under high pressure, a technique used in cryogenic electron microscopy and other scientific applications.

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Lab products found in correlation

Baf 400, by Oerlikon Balzers (2 mentions) Westase, by Takara Bio (2 mentions) Jem 1011 electron microscope, by JEOL (1 mentions) Ultracut uct, by Leica (1 mentions) Uc7 microtome, by Leica (1 mentions) Zymolyase 100t, by Nacalai Tesque (1 mentions) Protease inhibitor cocktail, by Nacalai Tesque (1 mentions) Bovine serum albumin (bsa), by Nacalai Tesque (1 mentions) Ccd camera, by Ametek (1 mentions) Afs 2 freeze substitution machine, by Leica (1 mentions) Epon araldite resin, by Electron Microscopy Sciences (1 mentions) Cryotube, by Thermo Fisher Scientific (1 mentions)

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HPM 010 (4 citations)

6 protocols using hpm 010 high pressure freezing machine

Ultrastructural Analysis of Caveolae

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Cells cultured on gold foils of 20 μm thickness were frozen using an HPM 010 high-pressure freezing machine (Leica). Freeze-fracture replicas prepared using a BAF400 apparatus (Baltec) were treated with SDS, labelled with mouse anti-CAV2 antibody (BD Bioscience) followed by colloidal gold (10 nm)-conjugated goat anti-mouse IgG antibody (British Biocell International) and observed under an JEM-1011 electron microscope (JEOL)22 (link)49 (link). The distribution density of the caveolar indentation was quantified using randomly taken electron micrographs. The areas of the plasma membranes in the respective micrographs were measured using ImageJ.
The cells cultured and unroofed as previously described were rapidly frozen by plunging them onto a copper block cooled with liquid nitrogen23 (link). The frozen samples were placed in the freeze-etching device (FR 9000, Hitachi), and the excess ice covering the samples was removed with pre-chilled glass knives before etching (slight freeze-drying). Etched surfaces were shadowed with platinum and carbon at −93 °C under vacuum at 5 × 10⁻⁶ Pa.

Yamaguchi T., Lu C., Ida L., Yanagisawa K., Usukura J., Cheng J., Hotta N., Shimada Y., Isomura H., Suzuki M., Fujimoto T, & Takahashi T. (2016). ROR1 sustains caveolae and survival signalling as a scaffold of cavin-1 and caveolin-1. Nature Communications, 7, 10060.

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In-resin CLEM Sample Preparation

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We followed a standard sample preparation protocol for in-resin CLEM (Kukulski et al., 2012 (link)). Immediately after barcoding, the yeast biomass was collected using a Millipore filtering setup on a 0.45-µm nitrocellulose filter. The cell slurry was transferred to the 0.1-mm-deep cavity of a 0.1/0.2-mm membrane carrier for an HPM010 high-pressure freezing machine or Leica ICE. The cavity was covered by the flat side of a 0.3-mm carrier, and the sandwich was inserted in the high-pressure freezing machine. Resin embedding was performed using a Leica AFS2 freeze-substitution machine equipped with a processing robot. Samples were embedded in Lowicryl HM20 resin using the freeze-substitution and embedding protocol optimized for in-resin CLEM (Kukulski et al., 2012 (link)). Dry acetone with 0.1% uranyl acetate was used as the freeze-substitution medium. The blocks were trimmed with a razor blade, and 100-nm-thick sections were produced using a Diatome 35° knife on a Leica Ultracut UCT or UC7 microtome. The sections were mounted on 200 mesh copper grids with continuous carbon support film (Electron Microscopy Sciences). Grids were imaged under the fluorescence microscope the same day.

Bykov Y.S., Cohen N., Gabrielli N., Manenschijn H., Welsch S., Chlanda P., Kukulski W., Patil K.R., Schuldiner M, & Briggs J.A. (2019). High-throughput ultrastructure screening using electron microscopy and fluorescent barcoding. The Journal of Cell Biology, 218(8), 2797-2811.

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Freeze Fracture Replica Preparation

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A copper EM grid (100 mesh) immersed with yeast or liposome pellets was sandwiched between a 20-µm-thick copper foil and a flat aluminum disc (242; Engineering Office M. Wohlwend) and frozen using an HPM 010 high-pressure freezing machine (Leica). For freeze fracture, the sandwiched sample was transferred to the stage of a Balzers BAF 400 and fractured at −115°C to −105°C under a vacuum of ∼10⁻⁶ mbar. Replicas were made by electron-beam evaporation in three steps: carbon (C; 5 nm in thickness) at an angle of 90° to the fractured surface, platinum-C (2 nm) at an angle of 45°, and C (10 nm) at an angle of 90°. The thickness of evaporation was adjusted by referring to a quartz crystal thickness monitor.
Replicas were treated sequentially with 2.5% SDS in 0.1 M Tris-HCl (pH 8.0) overnight at 60°C, with 0.1 mg/ml Zymolyase 100T (07665–55; Nacalai) in PBS containing 0.1% Triton X-100, 1% BSA (01281–26; Nacalai), and a protease inhibitor cocktail (25955–11; Nacalai) for 2 h at 37°C, and again with 2.5% SDS in PBS overnight at 60°C. In some experiments, the Zymolyase step was replaced with digestion with 0.5% Westase (9005; Takara Bio) in McIlvain citrate phosphate buffer (pH 6.0) containing 10 mM EDTA and 30% FCS (Tsuji et al., 2019 (link)). The replicas were stored in buffered 50% glycerol in PBS at −20°C until use.

Orii M., Tsuji T., Ogasawara Y, & Fujimoto T. (2021). Transmembrane phospholipid translocation mediated by Atg9 is involved in autophagosome formation. The Journal of Cell Biology, 220(3), e202009194.

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Yeast Freeze Fracture Electron Microscopy

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Freeze fracture EM analysis was performed as described previously^{28 (link)}. Briefly, yeast cells sandwiched between a 20-μm–thick copper foil and a flat aluminum disc (Engineering Office M. Wohlwend, Sennwald, Switzerland) were quick-frozen by high-pressure freezing using an HPM 010 high-pressure freezing machine according to the manufacturer’s instructions (Leica Microsystems, Wetzlar, Germany). Frozen specimens were transferred to the cold stage of a Balzers BAF 400 apparatus and fractured at − 120° under a vacuum of ~ 1 × 10⁻⁶ mbar. Freeze-fractured samples were subjected to a three step electron-beam evaporation: C (6 nm) at 90° Pt/C (2 nm) at 45°, and C (10 nm) as previously described^{28 (link)}. Thawed replicas were treated with 2.5% SDS in 0.1 M Tris–HCl (pH 8.0) at 60 °C overnight, with 0.1% Westase (Takara Bio, Kusatsu, Japan) in McIlvain buffer (37 mM citrate, 126 mM disodium hydrogen phosphate, pH 6.0) containing 10 mM EDTA, 30% fetal calf serum, and a protease inhibitor cocktail for 90 min at 30 °C, with 2.5% SDS again in 0.1 M Tris–HCl (pH 8.0) at 60 °C overnight. Replicas were observed and photographed with a JEOL JEM-1011 EM (Tokyo, Japan) using a CCD camera (Gatan, Pleasanton, CA, USA).

Kimura Y., Tsuji T., Shimizu Y., Watanabe Y., Kimura M., Fujimoto T, & Higuchi M. (2023). Physicochemical properties of the vacuolar membrane and cellular factors determine formation of vacuolar invaginations. Scientific Reports, 13, 16187.

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High-Pressure Freezing of Mouse Spleen Tissue

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Spleens from B6 and CD169^-/- mice were challenged retro-orbitally with FVC (2,500 SFFU, 5 dpi) isolated, divided into 8 equal pieces and immediately fixed with 3 % glutaraldehyde, 1 % paraformaldehyde, 5 % sucrose in 0.1 M sodium cacodylate trihydrate. Pre-fixed pieces of spleen were rinsed with fresh cacodylate buffer and placed individually into brass planchettes (Type A; Ted Pella, Redding, CA) prefilled with 10 % Ficoll in cacodylate buffer. The tissues were covered with the flat side of a Type-B brass planchette and rapidly frozen with a HPM-010 high-pressure freezing machine (Leica Microsystems, Vienna Austria). The frozen samples were transferred under liquid nitrogen to cryotubes (Nunc) containing a frozen solution of 2.5 % osmium tetroxide, 0.05 % uranyl acetate in acetone. Tubes were loaded into an AFS-2 freeze-substitution machine (Leica Microsystems) and processed at -90°C for 72 h, warmed over 12 h to -20°C, held at that temperature for 6 h, then warmed to 4°C for 2 h. The fixative was removed and the samples rinsed 4 x with cold acetone, following which they were infiltrated with Epon-Araldite resin (Electron Microscopy Sciences, Port Washington PA) over 48 h. The spleen tissue was flat-embedded between two Teflon-coated glass microscope slides. Resin was polymerized at 60°C for 48 h.

Uchil P.D., Pi R., Haugh K.A., Ladinsky M.S., Ventura J.D., Barrett B.S., Santiago M.L., Bjorkman P.J., Kassiotis G., Sewald X, & Mothes W. (2019). A Protective Role for the Lectin CD169/Siglec-1 against a Pathogenic Murine Retrovirus. Cell Host & Microbe, 25(1), 87-100.e10.

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High-Pressure Freezing and Freeze-Fracture Replica Formation

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Quick freezing and freeze-fracture replica formation were carried out as described previously [4 (link)]. In brief, samples sandwiched between a thin copper foil (20 μm) and a flat aluminum disc (Engineering Office M. Wohlwend, Sennwald, Switzerland) were frozen using an HPM 010 high-pressure freezing machine (Leica Microsystems, Wetzler, Germany), and freeze-fracture replicas were prepared in a BAF400 apparatus (BAL-TEC) by electron-beam evaporation in three steps: carbon (2–5 nm in thickness) at an angle of 90° to the specimen surface, platinum-carbon (1–2 nm) at an angle of 45°, and carbon (10–20 nm) at an angle of 90°. Thawed freeze-fracture replicas were treated with 2.5% SDS in 0.1 M Tris-HCl (pH 7.4) at 60°C overnight.

Aktar S., Takatori S., Tsuji T., Orii M., Ohsaki Y., Cheng J, & Fujimoto T. (2017). A New Electron Microscopic Method to Observe the Distribution of Phosphatidylinositol 3,4-bisphosphate. Acta Histochemica et Cytochemica, 50(5), 141-147.

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