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Hpm 010

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The HPM 010 is a high-performance microtome designed for precision cutting of tissue samples. It features a sturdy and reliable construction to ensure accurate and reproducible sectioning results.

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Lab products found in correlation

Baf 400, by Oerlikon Balzers (1 mentions) Ultracut uct, by Leica (1 mentions) Em afs2, by Leica (1 mentions) Afs2 chamber, by Leica (1 mentions) Tecnai t12, by Thermo Fisher Scientific (1 mentions) Hm 20, by Electron Microscopy Sciences (1 mentions) Afsii, by Leica (1 mentions) Cm120 transmission electron microscope, by Philips (1 mentions)

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HPM 010 high-pressure freezing machine (6 citations)

4 protocols using hpm 010

1

Ultrastructural Analysis of Autophagy in MEFs

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MEFs expressing GFP-LC3 were cultured in EBSS containing 0.25 mM propargylcholine for 1 h and quick-frozen using an HPM 010 high-pressure freezing machine (Leica). The frozen cells were freeze-fractured in a Balzers BAF 400 apparatus at −105 to −115 °C, and replicas were made by electron-beam evaporation22 (link). Thawed replicas were treated overnight with 2.5% SDS in PBS at 60 °C and subjected to label PC or GFP-LC3. For PC labeling, replicas were incubated for 30 min in reaction solution containing 5 μM biotin-azide (baseclick), 1 mM CuSO4, 100 μM Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, 2.5 mM ascorbic acid, 2% cold water fish skin gelatin, and 0.1 M Tris-HCl (pH 7.5), followed by incubation with anti-biotin antibody and then by colloidal gold (10 nm)-conjugated donkey anti-mouse IgG antibody21 (link). For labeling of GFP-LC3, replicas were incubated with rabbit anti-GFP antibody followed by colloidal gold (10 nm)-conjugated protein A. Labeled replicas were examined by electron microscope.
Ogasawara Y., Cheng J., Tatematsu T., Uchida M., Murase O., Yoshikawa S., Ohsaki Y, & Fujimoto T. (2020). Long-term autophagy is sustained by activation of CCTβ3 on lipid droplets. Nature Communications, 11, 4480.
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2

Ultrastructural Analysis of Synaptic Vesicle Docking

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Young adult worms were fixed by high-pressure freezing at -176 °C in the BAL-TEC HPM 010, and freeze substituted in Leica EM AFS2 with 2% osmium tetroxide and 0.1% uranyl acetate in acetone for 4 days at −90 °C and 16 hours at −20 °C. After infiltration and embedding in Durcupan ACM resin blocks were polymerized at 60 °C for 48 hours. Serial sections of 33 nm thicknesses were collected using Leica ULTRACUT UCT and stained for 5 minutes in 2.5% uranyl acetate in 70% methanol, followed by washing 3 minutes in Reynold’s lead citrate. Images of synapses from ventral nerve cord were obtained on a JEOL-1200 EX transmission electron microscope using Gatan 4 MP digital camera and DigitalMicrograph acquisition software. Distances from the edge of the dense projection to docked vesicles were measured using ImageJ. Distance from the dense projection to each docked vesicle was sorted into 33 nm bins. Number of vesicles in each bin was divided by number of profiles to yield an average number of vesicles per profile in each bin. Only vesicles in profiles containing a dense projection were included. Each contiguous set of serial profiles containing a dense projection was considered as a single synapse. Mean and SEM of all data points was used to calculate p values in two-tailed Student’s t test.
Zhou K., Cherra SJ I.I.I., Goncharov A, & Jin Y. (2017). Asynchronous cholinergic drive correlates with excitation-inhibition imbalance via a neuronal Ca2+ sensor protein. Cell reports, 19(6), 1117-1129.
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3

F. graminearum Microscopy Protocol

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For transmission electron microscopy (TEM) F. graminearum strain Tri4-RFP/Tri1-GFP was grown in MM and TIM for 65 h. Cultures were prepared by high pressure freezing (HPF) in a Balzers HPM 010 and freeze substitution (FS) in a Leica AFS2 chamber. Ultrathin microtome sections embedded in epoxy resin Poly/Bed 812 were imaged with microscope FEI Tecnai T12.
Boenisch M.J., Broz K.L., Purvine S.O., Chrisler W.B., Nicora C.D., Connolly L.R., Freitag M., Baker S.E, & Kistler H.C. (2017). Structural reorganization of the fungal endoplasmic reticulum upon induction of mycotoxin biosynthesis. Scientific Reports, 7, 44296.
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4

Yeast Sample Preparation for Electron Microscopy

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Yeast were prepared for electron microscopy by traditional chemical fixation using glutaraldehyde and paraformaldehyde followed by osmium staining as described previously [15 (link)]. High pressure freezing and freeze substitution was done according to previously described protocols [47 (link)]. Pelleted yeast were loaded into type-A planchettes and frozen in a Leica HPM-010. Freeze substitution was done in a Leica AFS II using substitution media containing: acetone, 0.25% glutaraldehyde, 0.05% UA, and 1% water. Samples were incubated in freeze substitution media at -80°C for 72 hours prior to warming to -20°C over 24 hours. Embedding was done using HM-20 (Electron Microscopy Sciences) over the course of several days at -20°C, starting with 25% HM-20 in acetone followed by 50%, 75%, and finally several washes of 100% HM-20. Each exchange was left for four hours to overnight. The resin was UV polymerized at -40°C for 48 hours followed by 12 hours of room temperature UV polymerization. All EM sections were post-stained with uranyl acetate and lead citrate [47 (link)]. Images were acquired on the Philips CM120 transmission electron microscope.
Sibert B.S., Navine A.K., Pennington J., Wang X, & Ahlquist P. (2018). Cowpea chlorotic mottle bromovirus replication proteins support template-selective RNA replication in Saccharomyces cerevisiae. PLoS ONE, 13(12), e0208743.
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