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Ultracut r microtome

Manufactured by Leica
Sourced in Austria, Germany

The Leica Ultracut R microtome is a high-precision instrument used for cutting thin sections of samples for microscopic examination. It features a motorized advancing mechanism and a user-friendly control panel for precise cutting and sample positioning.

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37 protocols using ultracut r microtome

1

Ultrastructure of Fish Olfactory Rosettes

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Olfactory rosettes were fixed with 2.5% glutaraldehyde solution (Sigma-Aldrich, USA) (prepared in 0.1 M phosphate buffer - PBS, pH 7.3) at 4 °C within 2 hours. After three washes in PBS, specimens were post-fixed in 2% OsO4 (Merck, Germany) in PBS for 12 hours, dehydrated in the ascending ethanol gradient with acetone, and encapsulated in resin (Araldite 502 Kit, SPI Supplies, USA). To estimate macro-anatomic features of fish olfactory rosettes of this preparations we made semi-thin sections (300–700 nm) with Ultracut R microtome (Leica, Germany). Stained with methylene blue (in a 1% aqueous solution, Sigma-Aldrich) that sections were investigated under an Axiovert 200 microscope (Carl Zeiss). Further, we obtained ultra-thin sections (70–80 nm) with the Ultracut R microtome (Leica, Germany). After staining with lead citrate, sections were analyzed with a transmission electron microscope Leo 906E at an accelerating voltage of 80 kV. Microscope images were taken with a MegaView II digital camera with the MegaVision software package (Soft Imaging System GmbH, Germany).
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2

Ultrastructural Analysis of Heat-Induced Rhabdomyolysis

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Immediately after heat stress, extensor digitorum longus (EDL) muscles were carefully dissected from CASQ1-null mice and fixed at room temperature in 3.5% glutaraldehyde 0.1 M Na cacodylate buffer, pH 7.2 overnight. Small bundles of fixed fibers were then postfixed in 2% OsO4 in the same buffer for 2 hrs and then block-stained in aqueous saturated uranyl acetate. After dehydration, specimens were embedded in an epoxy resin (Epon 812). Semithin (800 nm) histological sections were cut with a Leica Ultracut R Microtome (Leica Microsystem, Vienna, Austria) using a Diatome diamond knife (Diatome Ltd., Biel, Switzerland). After staining with toluidine blue dye, the sections were viewed using a Leica DMLB light microscope (Leica Microsystem, Vienna, Austria). The percentage of fibers presenting signs of rhabdomyolysis was determined as previously described [29 (link)].
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3

Ultrastructural Analysis of T-Tubule Network

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Intact soleus and EDL muscles were fixed at room temperature with 3.5 or 6% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) and processed for EM acquisition as previously described (Michelucci et al., 2019 (link)). For T-tubule staining, muscle samples were postfixed in a mixture of 2% OsO4 and 0.8% potassium ferrocyanide for 1–2 h followed by rinse with 0.1 M NaCaCo buffer with 75 mM CaCl2. Potassium ferrocyanide precipitate within the T-tubule network is visualized as an electron-dense dark precipitate in EM images (Boncompagni et al., 2017 (link)). Ultrathin sections (∼50 nm) were cut using a Leica Ultracut R microtome (Leica Microsystem) with a diamond knife (Diatome) and double-stained with uranyl acetate and lead citrate. Sections were viewed in a FP 505 Morgagni Series 268D electron microscope (FEI Company), equipped with Megaview III digital camera and Soft Imaging System at 60 or 100 kV (for T-tubule staining preparations).
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4

Ultrastructural Analysis of EDL Muscles

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EDL muscles were quickly dissected from sacrificed mice or rats, pinned on Sylgard dishes, fixed at room temperature with 3.5% glutaraldehyde in 0.1 M NaCaCO buffer (pH 7.2), and stored in the fixative at 4 °C before embedding. Fixed muscles were then post-fixed, embedded, stained en-block, as described previously [15 (link)]. For TT staining, specimens were post-fixed in a mixture of 2% OsO4 and 0.8% K3Fe (CN)6 for 1–2 h followed by a rinse with 0.1M NaCaCO buffer with 75 mM CaCl2. For histological examination, 700 nm thick sections were stained in a solution containing 1% Toludine blue O and 1% Sodium Borate Tetra in distilled water for 3 min on a hot plate at 55–60 °C. After washing and drying, sections were mounted with DPX mounting medium for Histology (Sigma-Aldrich, Milan, Italy) and observed with a Leica DMLB light microscope (Leica Microsystem, Vienna, Austria). For EM, ultrathin sections (~50 nm) were cut using a Leica Ultracut R microtome (Leica Microsystem, Vienna, Austria) with a Diatome diamond knife (Diatome, Biel, Switzerland) and double-stained with uranyl acetate and lead citrate. Sections were viewed in a FP 505 Morgagni Series 268D electron microscope (FEI Company, Brno, Czech Republic), equipped with Megaview III digital camera (Olympus Soft Imaging Solutions, Munster, Germany) and Soft Imaging System at 60 kV.
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5

Transmission Electron Microscopy of TiO2 NPs

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Briefly, TiO2 NP-treated SCs were fixed with 4% glutaraldehyde in 0.1 M sodium cacodylate (NaCaCO) buffer for 30 min and stored at 4°C. Cells were postfixed in 2% osmium tetroxide (OsO4) for 2 h and block stained in saturated uranyl acetate. After dehydration, specimens were embedded in epoxy resin (Epon 812), (Sigma-Aldrich Co., St. Louis, MO, USA). Ultrathin sections were cut using a Leica Ultracut R microtome (Leica Microsystem, Austria) with a Diatome knife (Diatome Ltd. CH-2501, Biel, Switzerland) and double stained with uranyl acetate replacement and lead citrate. Sections were viewed and photographed in a Morgagni Series 268D electron microscope (FEI Company, Brno, Czech Republic) equipped with a Megaview III digital camera and Analy-SIS software (Olympus Soft Imaging Solutions).
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6

Structural Analysis of CASQ1-Null Muscle

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EDL muscles were carefully dissected from WT and CASQ1-null mice at different ages (4 to 6, 14, 20, 24, and 27 months). Muscles were fixed at room temperature (RT) in 3.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, for 2 h and kept in fixative until further use. Small bundles of fibers were then post-fixed and embedded as described by Paolini et al. [25 (link)]. For histological analyses, longitudinal and cross-oriented semithin sections (250 nm) were cut with a Leica Ultracut R microtome (Leica Microsystem, Vienna, Austria) using a Diatome diamond knife (Diatome Ltd. CH-2501 Biel, Switzerland). After staining with toluidine blue dye, the sections were viewed on a Leica DMLB fluorescence microscope (Leica Microsystem, Vienna, Austria). For electron microscopy (EM), ultrathin sections were cut (approximately 50 nm), stained in 4% uranyl acetate and lead citrate, and examined with a Morgagni Series 268D electron microscope (FEI Company, Brno, Czech Republic) equipped with a Megaview III digital camera.
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7

Histological Analysis of Muscle Fibers

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Cryosections of both P12 newborns hindlimb cross-sections and adult TA were stained for H&E and SDH. The total myofiber numbers and CSA in conditional model were calculated from entire hindlimb cross-section based on assembled mosaic image (×20 magnification). Adult mice CSA was performed on TA as described29 (link). For EM, EDL muscles were dissected from killed animals, pinned on a Sylgard dish, fixed at room temperature with 3.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (NaCaCO) (pH 7.4), and stored in the fixative at 4 °C. Fixed muscles were then postfixed in a mixture of 2% OsO4 and 0.8% K3Fe(CN)6 for 1–2 h, rinsed with 0.1 M NaCaCO with 75 mM CaCl2, en-block stained with saturated uranyl acetate, and embedded for EM in epoxy resin (Epon 812) as in ref. 48 (link). Ultrathin sections (~40 nm) were cut in a Leica Ultracut R microtome (Leica Microsystem, Austria) using a Diatome diamond knife (DiatomeLtd. CH-2501 Biel, Switzerland) and examined at 60 kV after double-staining with uranyl acetate and lead citrate, with a FP 505 Morgagni Series 268D electron microscope (FEI Company, Brno, Czech Republic), equipped with Megaview III digital camera (Munster, Germany) and Soft Imaging System (Germany).
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8

Perfusion and Ultrastructural Analysis of Fish Sonic Muscles

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For perfusion, fish were deeply anesthetized by immersion in 0.025% benzocaine in seawater. Morphometric measurements (body mass and length) were taken, and fish were then perfused transcardially with teleost Ringer’s solution followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2. The swimbladders with their attached muscles were dissected out and stored in 4% paraformaldehyde in 0.1 M phosphate buffer. Testes were also dissected out and weighed. Subsequently, a portion of each sonic muscle was dissected off of each excised bladder, post-fixed in 4% paraformaldehyde with 2.5% glutaraldehyde, and either used immediately for embedding, or shipped to Italy (to SB) for electron microscopy.
For E.M. embedding, the muscles were further post-fixed in 2% OsO4 in 0.1 M cacodylate buffer pH 7.2, washed in 0.1 M acetate buffer pH 5.4, en-bloc stained in 2% aqueous uranyl acetate for 1 h, dehydrated and embedded in Epon. Thin (∼80 nm) sections were cut using a Leica Ultracut R microtome (Leica Microsystem) with a Diatome diamond knife (Diatome Ltd.) and double-stained with uranyl acetate in 50% EtOH for 5 min and then 5 min in Sato’s lead solution.
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9

Electron Microscopy of Muscle Tissues

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For EM, EDL and Soleus muscles were dissected from sacrificed animals, pinned on a Sylgard dish, fixed at room temperature with 3.5% glutaraldehyde in 0.1 M NaCaCO buffer (pH 7.4), and stored in the fixative at 4°C. Fixed muscles were then post‐fixed in a mixture of 2% OsO4 and 0.8% K3Fe(CN)6 for 1–2 h, rinsed with 0.1 M sodium cacodylate buffer with 75 mM CaCl2, en‐block stained with saturated uranyl acetate, and embedded for EM in epoxy resin (Epon 812). Ultrathin sections (~40 nm) were cut in a Leica Ultracut R microtome (Leica Microsystem, Austria) using a Diatome diamond knife (DiatomeLtd. CH‐2501 Biel, Switzerland) and examined at 60 kV after double staining with uranyl acetate and lead citrate, with a FP 505 Morgagni Series 268D electron microscope (FEI Company, Brno, Czech Republic), equipped with Megaview III digital camera (Munster, Germany) and Soft Imaging System (Germany).
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10

Ultrastructural Analysis of Regenerating Plant Protoplasts

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The protocol for cryofixation, freeze-substitution, sectioning, immunolabeling, and viewing on the TEM followed Wilson and Bacic (2012), with minor alterations. A Leica EMPACT2 high pressure freezer (Leica Microsystems) was used to cryofix protoplasts at a concentration of 1 × 105 protoplasts/mL at a 0, 1, 2, 4, and 24 h regeneration time and 7-day old cells. A Leica AFS2 freeze substitution unit (Leica Microsystems) was used for freeze substitution with 0.1% uranyl acetate in acetone for 48 h at −90 °C, before warming up to −50 °C. The samples were washed in acetone at −50 °C, followed by low temperature embedding in Lowicryl HM20 (Electron Microscopy Sciences). Thin sections were cut on a Leica Ultracut R microtome (Leica Microsystems), followed by immunolabeling, as described above. Images were taken using either a Philips CM120 BioTWIN or a Tecnai G2 Spirit transmission electron microscope (Thermofisher Scientific, formerly FEI).
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