Lesions were excised with a surgical scissors after 20 days of treatment (50 days post infection), washed in PBS and fixed in 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer pH 7.2 with 3.5% sucrose for 1h/4 °C. Samples were post-fixed with 1% OsO4 (1 h/4 °C) in cacodylate buffer and, after washing, the samples were dehydrated in serial acetone concentrations (30%, 50%, 70%, 90% and 100%). Finally, samples were embedded in PolyBed 812 resin and polymerized at 72 h/ 60 °C. After polymerization, semi-thin sections were made in an Ultracut S ultramicrotome (Leica, Vienna, Austria) stained with toluidine blue and eosin and analyzed under a light microscope - Axio imager 2 (Zeiss, Göttingen, Germany). After selecting the areas of interest, ultra-thin sections were obtained in Leica Ultracut S ultramicrotome, collected in 300 mesh copper grids, contrasted with 5% and lead citrate and analyzed in the transmission electron microscope - JEOL JEM-1011 (Boston, MA, USA).
Ultracut s ultramicrotome
Manufactured by Leica
Sourced in Germany, Austria, United States, Switzerland
The Leica Ultracut S is an ultramicrotome designed for the precision cutting of ultra-thin sections for electron microscopy. It features a high-precision feed mechanism and a robust design for reliable operation.
Automatically generated - may contain errors
Lab products found in correlation
Eponate 12 resin, by Ted Pella (9 mentions)
Diamond knife, by Electron Microscopy Sciences (7 mentions)
Jem 1400 tem, by JEOL (6 mentions)
Jem 1400 electron microscope, by JEOL (5 mentions)
Gatan us1000 2k 2k ccd camera, by Ametek (4 mentions)
Jem 1400 transmission electron microscope, by JEOL (3 mentions)
Us1000 ccd camera, by Ametek (3 mentions)
Diatome diamond knife, by Electron Microscopy Sciences (3 mentions)
Eponate 12, by Ted Pella (2 mentions)
Jem 1011, by JEOL (2 mentions)
Item software, by Olympus (2 mentions)
Vt1000, by Leica (2 mentions)
Jem 1400 transmission electron microscope, by Ametek (2 mentions)
Osmium tetroxide, by Merck Group (2 mentions)
Glutaraldehyde, by Merck Group (2 mentions)
Veleta 2k 2k digital camera, by Olympus (2 mentions)
Em900 electron microscope, by Zeiss (2 mentions)
Thiocarbohydrazide, by Thermo Fisher Scientific (2 mentions)
Technovit 8100 resin, by Kulzer (2 mentions)
Jem 1400 lab6 transmission electron microscope, by JEOL (2 mentions)
76 protocols using ultracut s ultramicrotome
1
Ultrastructural Analysis of Lesion Samples
2
Ultrathin Section Staining and Imaging
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Ultrathin sections (50 nm, Leica Ultracut S ultramicrotome) were stained with aqueous 4 % uranyl acetate and lead citrate. Electron micrographs were obtained as outlined above [28 (link), 49 (link)].
3
Glycerol-Induced Morphological Changes in V. alginolyticus
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To observe the morphological changes related to cell growth on glycerol as the carbon source, V. alginolyticus strains LHF01 and LHF02 were cultured in TYS medium supplemented with 20 g/L glycerol for 24 h for TEM observation. The strains cultivated in TYS medium without an additional carbon source were used as the control group. The cell culture was centrifuged to collect the bacterial pellet, washed 1–2 times with PBS and mixed with a pre-cooled fixative solution, and stored at 4 °C for at least 12 h. The fixative solution was poured out, then the samples were rinsed 3 times with a 0.1 M, pH7.0 phosphate buffer. Then, the samples were fixed with a 1% osmium acid solution for 2 h, rinsed 3 times with 0.1 M, pH7.0 phosphate buffer, and dehydrated with graded ethanol solutions and acetone. The samples were then embedded in a mixture of embedding agent and acetone, sliced using a Leica Ultracut S ultramicrotome, and stained with a lead citrate solution and a uranyl acetate 50% ethanol saturated solution. The prepared sample was visualized using a JEM-2100F transmission electron microscope (JEOL Ltd., Tokyo, Japan).
4
Ultrastructural Analysis of PEITC-Treated Cells
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MNNG/HOS OS cells were seeded in 6-well cell culture plates with a density of 1 × 105 cells/mL and allowed to attach. The following day, cells were treated with 30 μM PEITC for 4 h, 12 h, and 24 h, respectively. Cells were collected and washed by PBS, fixed in 2% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer before washing in 0.1 M sodium cacodylate buffer, then followed by postfixing in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer. After fixation, cells were dehydrated through a graded series of alcohols and embedded in Spurrs Resin. Ultrathin sections were cut with a diamond knife using a Leica Ultracut S ultra-microtome and stained with both methanolic uranyl acetate and lead citrate before viewing in a LEO 906 transmission electron microscope operated at 60 kV.
5
Spinal Cord Sectioning and Staining for Myelin Visualization
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The spinal cord was sectioned coronally using a vibratome (Leica Biosystems) to prepare samples for semi-thin sections. 20% gelatin was prepared by dissolving gelatin powder (Merck Millipore; 1040781000) in PBS by stirring and heating at 60°C. Before vibratome sectioning, the aliquots of 20% gelatin were thawed from −20°C and heated at 37°C with shaking. A segment of spinal cord (shorter than 3 mm) was freshly dissected from the mouse and embedded in 20% gelatin in an embedding mold (Sigma; E4140-1EA) on ice. 200-µm coronal sections were cut at a speed of 0.4–0.5 mm/s. The coronal sections were fixed with a solution containing 4% PFA and 2.5% glutaraldehyde in Karlsson & Schultz buffer, under a coverslip in a 24-well plate at 4°C until further processing. The spinal cord sections were embedded in epon and cut at a thickness of 500 nm using the Leica Ultracut S ultramicrotome. Semi-thin sections were stained with methylene blue-azure II to visualize the lipid-rich areas such as myelin.
6
Ultrastructural Analysis of Radioles
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For TEM, radioles of several individuals of each species were fixed in 2.5% glutaraldehyde buffered in 0.05 M phosphate buffer with 0.3 M NaCl (2 to 24 hours at 4°C). Ruthenium red (~0.5%) was added to the fixative. The specimens were rinsed in the same buffer and postfixed with 1% OsO4 for 30 min. The radioles were dehydrated in an ascending alcohol series and embedded in Spurr’s resin. Silver interference–colored sections (65 to 70 nm) were prepared using a diamond knife (Diatome Ultra 45°) on a Leica Ultracut S ultramicrotome. The sections were placed on Formvar-covered, single-slot copper grids and stained with 2% uranyl acetate and lead citrate in an automated TEM stainer (QG-3100, Boeckeler Instruments). The sections were examined using a Zeiss EM10 transmission electron microscope with digital imaging plates (DITABIS Digital Biomedical Imaging Systems, Germany).
7
Tissue Preparation for TEM Imaging
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Tissue was fixed in 2 % paraformaldehyde, 2.5 % glutaraldehyde in 0.08 M Sorensen’s phosphate buffer (PBS) for 2 h and washed in PBS before being immersed in 2 % osmium tetroxide in PBS and dehydrated through a graded series of alcohols, two acetone rinses and embedding in Spurrs resin. Sections approximately 80 nm thick were cut with a diamond knife (Diatome, Switzerland) on an Ultracut-S ultramicrotome (Leica, Mannheim, Germany) and contrasted with uranyl acetate and lead citrate. Images were captured with a Megaview II cooled CCD camera (Soft Imaging Solutions, Olympus) using a JEOL 1011 transmission electron microscope (TEM).
8
Photooxidation-mediated GFP Neuron Labeling for EM
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Photooxidation-mediated specific labeling of GFP-expressing neurons for EM was done as described by Horstmann et al. (2013 (link)). Briefly, perfusion-fixed (4% PFA) brain slices from animals injected with AAV-synaptophysin-2-pHluorin were cut at 200 μm thickness. The slice of interest was incubated in an oxygenated Tris-HCl buffer overnight. The following day, the pHluorin was excited under a light microscope in a 1 mg/mL 3,3′-Diaminobenzidine (DAB) supplemented Tris-HCl buffer. The subsequently excised region of interest with the DAB-precipitate was embedded in epoxy resin. For EM the tissue block was serially sectioned at a thicknes of 38 nm using an Ultracut S ultramicrotome (Leica, Germany) equipped with a diamond knife angled at 35° (Diatome, Biel, Germany). The 109 sections were collected onto hydrophilized silicon wafer strips and post stained with Reynolds lead citrate.
9
Ultrastructural Analysis of APR-246 Treatment
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FLO-1 and JH-EsoAd1 cells were seeded in 10 cm dishes. The following day, cells were treated with vehicle (H2O) or APR-246 (FLO-1: 25 μM and JH-EsoAd1: 40 μM) for 15 and 24 h. Cells were collected in PBS, gently spun down, fixed in 2% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer before washing in 0.1 M sodium cacodylate buffer and post-fixing in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer. Following fixation, cells were dehydrated through a graded series of alcohols and embedded in Spurrs Resin. Ultrathin sections were cut with a diamond knife using a Leica Ultracut S ultra-microtome (Austria), stained with both methanolic uranyl acetate and lead citrate before viewing in a JEOL 1011 transmission electron microscope (Japan) at 60 kV. Images were recorded with a MegaView III CCD cooled digital camera (Soft Imaging Systems, Germany) at × 10,000– × 80,000 magnification. At least 10 mitochondria were examined per treatment group. Representative images are shown.
10
Detailed Sciatic Nerve Imaging Protocol
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This method has been described [39 (link)]. Epon sections (1μM thickness) of mouse sciatic nerves were examined under the 63X objective. The entire field of transverse sections of each nerve was imaged for analysis. Images were imported into software (ImagePro Plus). Areas of each field were counted to obtain the number of nerve fibers.
Electron microscopy on mouse sciatic nerves was performed as described [40 (link)]. Briefly, sciatic nerves were cryofixed in a high-pressure freezer (HPM100; Leica) and freeze substitution was performed in an embedding system at low temperature (AFS; Leica) using the tannic acid–OsO4 protocol. Samples were embedded in Epon, sectioned (Ultracut S Ultramicrotome, Leica), and stained with an aqueous solution of 2% uranyl acetate followed by lead citrate. Samples were examined in a LEO EM 912AB electron microscope (Zeiss, Oberkochen, Germany). Pictures were taken with an on-axis 2048x2048-CCD-camera (TRS, Moorenweis, Germany).
Electron microscopy on mouse sciatic nerves was performed as described [40 (link)]. Briefly, sciatic nerves were cryofixed in a high-pressure freezer (HPM100; Leica) and freeze substitution was performed in an embedding system at low temperature (AFS; Leica) using the tannic acid–OsO4 protocol. Samples were embedded in Epon, sectioned (Ultracut S Ultramicrotome, Leica), and stained with an aqueous solution of 2% uranyl acetate followed by lead citrate. Samples were examined in a LEO EM 912AB electron microscope (Zeiss, Oberkochen, Germany). Pictures were taken with an on-axis 2048x2048-CCD-camera (TRS, Moorenweis, Germany).
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