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).
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.
Automatically generated - may contain errors
Lab products found in correlation
Fp 505 morgagni series 268d electron microscope, by Thermo Fisher Scientific (9 mentions)
Megaview 3 digital camera, by Thermo Fisher Scientific (6 mentions)
Megaview 3 digital camera, by Olympus (3 mentions)
Morgagni series 268d electron microscope, by Thermo Fisher Scientific (3 mentions)
Cm12 tem, by Ametek (3 mentions)
Dmlb light microscope, by Leica (2 mentions)
Diatome diamond knife, by Electron Microscopy Sciences (2 mentions)
Diamond knife, by Electron Microscopy Sciences (2 mentions)
Jem 1400 flash transmission electron microscope, by JEOL (2 mentions)
Araldite 502 kit, by SPI Supplies (2 mentions)
Dpx mounting medium for histology, by Merck Group (1 mentions)
Epon 812, by Merck Group (1 mentions)
Dmlb fluorescence microscope, by Leica (1 mentions)
Tecnai g2 spirit, by Thermo Fisher Scientific (1 mentions)
Lowicryl hm20, by Electron Microscopy Sciences (1 mentions)
Oneview camera, by Ametek (1 mentions)
Jem arm200f, by JEOL (1 mentions)
Epon 812 resin, by Electron Microscopy Sciences (1 mentions)
H7500 tem, by Hitachi (1 mentions)
Glutaraldehyde, by Electron Microscopy Sciences (1 mentions)
37 protocols using ultracut r microtome
1
Ultrastructure of Fish Olfactory Rosettes
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)].
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).
4
Ultrastructural Analysis of EDL Muscles
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).
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.
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).
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.
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.
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).
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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