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Ultracut e microtome

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The Ultracut E microtome is a laboratory equipment designed for precision sectioning of biological and materials samples. It is capable of producing thin sections with thicknesses ranging from 50 nanometers to 2 micrometers. The device utilizes a motorized cutting action to generate high-quality tissue sections for microscopic analysis.

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Lab products found in correlation

Diamond knife, by Electron Microscopy Sciences (7 mentions) Lead citrate, by Leica (1 mentions) Osmium tetroxide, by Electron Microscopy Sciences (1 mentions) Uranyl acetate, by Merck Group (1 mentions) Epon 812, by Merck Group (1 mentions) Tecnai spirit tem, by Thermo Fisher Scientific (1 mentions) 0.1 m sodium cacodylate buffer, by Merck Group (1 mentions) X maxn 80 tle eds, by Oxford Instruments (1 mentions) Jem 2010f tem, by JEOL (1 mentions) Embed 812 resin, by Electron Microscopy Sciences (1 mentions) H7600 tem, by Hitachi (1 mentions) Lr white, by SPI Supplies (1 mentions) Jem 2100f, by JEOL (1 mentions)

14 protocols using ultracut e microtome

1

Transmission Electron Microscopy of Cells

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Cells were fixed in 2.5% gluteraldehyde, 3% PFA with 5% sucrose in 0.1 M sodium cacodylate buffer (pH 7.4), pelletted and post fixed in 1% OsO4 in veronal-acetate buffer. The cell pellet was stained in block overnight with 0.5% uranyl acetate in veronal-acetate buffer (pH 6.0), then dehydrated and embedded in Embed-812 resin. Sections were cut on a Reichert Ultracut E microtome with a Diatome diamond knife at a thickness of 50 nm, stained with uranyl acetate and lead citrate. The sections were examined using an FEI Tecnai spirit at 80 KV and photographed with an AMT CCD (charge-coupled device) camera.
Connor Y., Tekleab S., Nandakumar S., Walls C., Tekleab Y., Husain A., Gadish O., Sabbisetti V., Kaushik S., Sehrawat S., Kulkarni A., Dvorak H., Zetter B., R. Edelman E, & Sengupta S. (2015). Physical nanoscale conduit-mediated communication between tumour cells and the endothelium modulates endothelial phenotype. Nature Communications, 6, 8671.
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2

Ultrastructural Tissue Analysis Protocol

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Animals were kept on ice for 10 min before fixation with cold 2.5% glutaraldehyde, 3% paraformaldehyde with 5% sucrose in 0.1 M sodium cacodylate buffer (pH 7.4) overnight, then post-fixed in 1% OsO4 in veronal-acetate buffer. Animals were stained overnight with 0.5% uranyl acetate in veronal-acetate buffer (pH 6.0), dehydrated, and embedded in Spurr's resin. Sections were cut on a Reichert Ultracut E microtome with a Diatome diamond knife at a thickness setting of 50 nm then stained with 2% uranyl acetate and lead citrate. The sections were examined using a FEI Tecnai spirit at 80 KV and photographed with an AMT CCD camera.
Cote L.E., Simental E, & Reddien P.W. (2019). Muscle functions as a connective tissue and source of extracellular matrix in planarians. Nature Communications, 10, 1592.
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3

Electron Microscopy Fixation and Imaging of Erythrocytes

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For cell EM, cell fixative was first prepared as 2.5% gluteraldehyde, 3% paraformaldehyde with 5% sucrose in 0.1M sodium cacodylate buffer (pH 7.4). Since erythrocytes are in suspension, 200 µL of fixative was added to 200 µL sample volume, rotated for 1 min at incubation temperature of 37°C, pelleted, and resuspended in 1 mL fixative. After fixing, cells were pelletted, and post-fixed in 1% OsO4 in veronal-acetate buffer. The cell pellet was stained in block overnight with 0.5% uranyl acetate in veronal-acetate buffer (pH 6.0), then dehydrated and embedded in Embed-812 resin. Sections were cut on a Reichert Ultracut E microtome with a Diatome diamond knife at a thickness setting of 50 nm. The sections were examined using a FEI Tecnai Spirit at 80KV and photographed with an AMT CCD camera.
Atukorale P.U., Yang Y.S., Bekdemir A., Carney R.P., Silva P.J., Watson N., Stellacci F, & Irvine D.J. (2015). Influence of the Glycocalyx and Plasma Membrane Composition on Amphiphilic Gold Nanoparticle Association with Erythrocytes. Nanoscale, 7(26), 11420-11432.
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4

Electron Microscopy of HT-29 and LoVo Cells

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HT-29 and LoVo grown and treated as decribed above on inserts (BD Falcon, Milan Dutscher Group, Nesselnbach/Niederwil, Switzerland, #353090) in six-well-plates were processed for electron microscopy as follows: Each insert was fixed in a solution of 3% Glutaraldehyde (25% EM Grade, Agar Scientific, Stansted, UK, AGR1010) in 0.1 M potassium phosphate buffer (total osmolarity 630 mOsm, pH 7.2). Thereafter the inserts were postfixed in a 1% solution of Osmium Tetroxide (Electron Microscopy Sciences, Hatfield, USA) in 0.1 M sodium cacodylate buffer (total osmolarity 356 mOsm, pH 7.2) and dehydrated in increasing concentrations of ethanol, 70%, 80%, 96% and 100%. The membranes of the inserts were cut out and embedded in Epon 812 (Sigma Aldrich, Wien, Austria, 45345-250ML-F). Ultrathin sections of 70 nm were cut in flat angle on a Reichert-Jung Ultracut E Microtome using a diamond knife (Diatome, Biel, Switzerland), put on 200 mesh hexagonal copper grids (Plano, Wetzlar, Germany) and double stained with 1% Uranyl Acetate (Sigma Aldrich, Wien, Austria, 73943) and 3% Lead Citrate (Leica Microsystems, Heerbrugg, Switzerland, 16705530). Electron micrographs were taken on a Morgagni M268 Electron Microscope (FEI, Brno, Czech Republic).
Niklaus M., Adams O., Berezowska S., Zlobec I., Graber F., Slotta-Huspenina J., Nitsche U., Rosenberg R., Tschan M.P, & Langer R. (2017). Expression analysis of LC3B and p62 indicates intact activated autophagy is associated with an unfavorable prognosis in colon cancer. Oncotarget, 8(33), 54604-54615.
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5

Ultrastructural Analysis of hESCs

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Cells (hESCs) were fixed in 2.5 % glutaraldehyde, 3 % paraformaldehyde with 5 % sucrose in 0.1 M sodium cacodylate buffer (pH 7.4) (Sigma-Aldrich), pelleted, and post-fixed in 1 % OsO4 in veronal-acetate buffer. The cell pellet was stained in block overnight with 0.5 % uranyl acetate in veronal-acetate buffer (pH 6), then dehydrated and embedded in Embed-812 resin. Sections were cut on Reichert Ultracut-E microtome with a Diatome diamond knife at 50 nm thickness setting, stained with uranyl acetate and lead citrate, then examined using Tecnai Spirit TEM (FEI) at 80 KV and photographed with AMT CCD camera.60 (link)
Sun C., Seranova E., Cohen M.A., Chipara M., Roberts J., Astuti D., Palhegyi A.M., Acharjee A., Sedlackova L., Kataura T., Otten E.G., Panda P.K., Lara-Reyna S., Korsgen M.E., Kauffman K.J., Huerta-Uribe A., Zatyka M., Silva L.F., Torresi J., Zhang S., Hughes G.W., Ward C., Kuechler E.R., Cartwright D., Trushin S., Trushina E., Sahay G., Buganim Y., Lavery G.G., Gsponer J., Anderson D.G., Frickel E.M., Rosenstock T.R., Barrett T., Maddocks O.D., Tennant D.A., Wang H., Jaenisch R., Korolchuk V.I, & Sarkar S. (2023). NAD depletion mediates cytotoxicity in human neurons with autophagy deficiency. Cell reports, 42(5), 112372.
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6

Electron Microscopy Sample Preparation

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Cells were fixed in 2.5% gluteraldehyde, 3% PFA with 5% sucrose in 0.1 M sodium cacodylate buffer (pH 7.4), pelletted and post fixed in 1% OsO4 in veronal-acetate buffer. The cell pellet was stained in block overnight with 0.5% uranyl acetate in veronal-acetate buffer (pH 6.0), then dehydrated and embedded in Embed-812 resin. Sections were cut on a Reichert Ultracut E microtome with a Diatome diamond knife at a thickness of 50 nm, stained with uranyl acetate and lead citrate. The sections were examined using an FEI Tecnai spirit at 80 KV and photographed with an AMT CCD (charge-coupled device) camera.
Connor Y., Tekleab S., Nandakumar S., Walls C., Tekleab Y., Husain A., Gadish O., Sabbisetti V., Kaushik S., Sehrawat S., Kulkarni A., Dvorak H., Zetter B., R. Edelman E, & Sengupta S. (2015). Physical nanoscale conduit-mediated communication between tumour cells and the endothelium modulates endothelial phenotype. Nature Communications, 6, 8671.
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7

Ultrastructural Analysis of Differentiated β-like Cells

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Differentiated Stage 7 β-like cells were fixed in 2.5% glutaraldehyde, 3% paraformaldehyde with 5% sucrose in 0.1M sodium cacodylate buffer (pH 7.4), pelleted, and post fixed in 1% OsO4 in veronal-acetate buffer. The cells were stained overnight with 0.5% uranyl acetate in veronal-acetate buffer (pH 6.0), then dehydrated and embedded in Embed-812 resin. Sections were cut on a Reichert Ultracut E microtome with a Diatome diamond knife at a thickness setting of 50 nm, stained with uranyl acetate, and lead citrate. The sections were examined using a FEI Tecnai spirit at 80KV and photographed with an AMT CCD camera.
Ma H., Jeppesen J.F, & Jaenisch R. (2020). Human T Cells Expressing a CD19 CAR-T Receptor Provide Insights into Mechanisms of Human CD19-Positive β Cell Destruction. Cell Reports Medicine, 1(6), 100097.
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8

Nanoparticle Detection in Tissue Samples

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Sample preparation procedures for detecting NPs have been reported in our recent publication (Zhao et al., 2016a (link),b (link); Ge et al., 2017 (link); Liu et al., 2017 (link)). Briefly, tissue samples were collected and fixed for 2 h in 2% glutaraldehyde made in sodium phosphate buffer (pH 7.2). Specimens were then washed extensively to remove the excess fixative and subsequently post-fixed in 1% OsO4 for 1 h in the dark. Specimens were then dehydrated in an increasingly graded series of ethanol and infiltrated with increased concentrations of Spur’s embedding medium in propylene epoxide. Subsequently, the specimens were polymerized in embedding medium for 12 h at 37°C, 12 h at 45°C, and 48 h at 60°C. Fifty nanometer sections were cut on a Leica Ultracut E microtome equipped with a diamond knife (Diatome, Hatfield, PA, United States), and collected on form var-coated, carbon-stabilized Mo grids. The section containing grids were stained with uranyl acetate, air dried overnight, and imaged on a JEM-2010F TEM (JEOL Ltd., Japan). The presence of ZnO NPs in the tissues was confirmed by using X-MaxN 80 TLE EDS (Oxford Instruments, United Kingdom).
Zhang W., Zhao Y., Li F., Li L., Feng Y., Min L., Ma D., Yu S., Liu J., Zhang H., Shi T., Li F, & Shen W. (2018). Zinc Oxide Nanoparticle Caused Plasma Metabolomic Perturbations Correlate with Hepatic Steatosis. Frontiers in Pharmacology, 9, 57.
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9

Ultrastructural Analysis of Ventral Bodywall Muscles

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Animals were kept on ice for 10 min before fixation with cold 2.5% glutaraldehyde, 3% paraformaldehyde with 5% sucrose in 0.1 M sodium cacodylate buffer (pH 7.4) overnight, then post-fixed in 1% OsO4 in veronal-acetate buffer. Animals were stained overnight with 0.5% uranyl acetate in veronal-acetate buffer (pH 6.0), dehydrated, and embedded in Spurr’s resin. Transverse sections were cut on a Reichert Ultracut E microtome with a Diatome diamond knife at a thickness setting of 50 nm then stained with 2% uranyl acetate and lead citrate. The sections were examined using a FEI Tecnai spirit at 80 KV and photographed with an AMT CCD camera. All images were taken on the ventral BWM at 6800X. Muscle fibers were traced by hand and pseudocolored by the fiber orientation, size, and distance from the subepidermal membrane. Circular fibers were defined as the outermost layer adjacent to the subepidermal membrane with myosin fibers running sagittal to the plane of section, and pseudocolor in magenta. Longitudinal fibers on the ventral side were thick, and myosin fibers were transversal to the plane of section and pseudocolored in green. All other identifiable muscle fibers were pseudocolored in yellow. For visualization ease, images were blurred with ImageJ’s smooth function.
Scimone M.L., Cote L.E, & Reddien P.W. (2017). Orthogonal muscle fibers have different instructive roles in planarian regeneration. Nature, 551(7682), 623-628.
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10

Electron Microscopy Sample Preparation

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Cells were fixed in 2.5% glutaraldehyde, 3% paraformaldehyde with 5% sucrose in 0.1 M sodium cacodylate buffer (pH 7.4), pelleted, and post-fixed in 1% OsO4 in veronal-acetate buffer. The cell pellet was stained en bloc overnight with 0.5% uranyl acetate in veronal-acetate buffer (pH 6.0), then dehydrated and embedded in Embed-812 resin.
Sections were cut on a Leica Ultracut E microtome with a Diatome diamond knife at a thickness setting of 50 nm, stained with uranyl acetate, and lead citrate. The sections were examined using a FEI Tecnai spirit at 80 kV and photographed with an AMT CCD camera.
Keckesova Z., Donaher J.L., De Cock J., Freinkman E., Lingrell S., Bachovchin D.A., Bierie B., Tischler V., Noske A., Okondo M.C., Reinhardt F., Thiru P., Golub T.R., Vance J.E, & Weinberg R.A. (2017). LACTB is a tumour suppressor that modulates lipid metabolism and cell state. Nature, 543(7647), 681-686.
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