The cells were fixed in 1× phosphate buffered saline (PBS) containing 3% glutaraldehyde. After washing in 0.1 M Soerensen’s phosphate buffer (Merck, Darmstadt, Germany), the samples were post-fixed in 1% osmium tetroxide (OsO4) (Roth, Karlsruhe, Germany) solved in 25 mM sucrose buffer (Merck) and dehydrated by ascending ethanol series (30%, 50%, 70%, 90%, and 100%) for 10 min each. The last step was repeated three times. Subsequently, dehydrated specimens were incubated in propylene oxide (Serva, Heidelberg, Germany) for 30 min, in a mixture of Epon resin (Serva) and propylene oxide (1:1) for 1 h, and finally, in pure Epon for 1 h. Epon polymerization was performed at 90 °C for 2 h. Finally, ultrathin sections (70–100 nm) were cut with an ultramicrotome (Reichert Ultracut S, Leica, Wetzlar, Germany) using a diamond knife (Diatome Ltd., Nidau, Switzerland) and picked up on Cu/Rh grids (HR23 Maxtaform, Plano GmbH, Wetzlar, Germany). Contrast was enhanced by staining with 0.5% uranyl acetate and 1% lead citrate (both Science Services, Munich, Germany). Samples were viewed at an acceleration voltage of 60 kV using a Zeiss Leo 906 (Carl Zeiss AG, Oberkochen, Germany) transmission electron microscope. Pictures were acquired at magnifications of 6000× to 100,000×.
Uranyl acetate
Manufactured by Science Services
Sourced in Germany
Uranyl acetate is a chemical compound with the formula UO2(CH3COO)2. It is a yellow crystalline solid that is soluble in water and other polar solvents. Uranyl acetate is commonly used as a staining agent in electron microscopy, where it is used to enhance the contrast of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Lead citrate, by Science Services (5 mentions)
Leo 906, by Zeiss (3 mentions)
Sucrose buffer, by Merck Group (2 mentions)
Propylene oxide, by Serva Electrophoresis (2 mentions)
Soerensen s phosphate buffer, by Merck Group (2 mentions)
Epon resin, by Serva Electrophoresis (2 mentions)
Lead citrate, by Merck Group (2 mentions)
Reichert ultracut s, by Leica (1 mentions)
Copper grid, by Science Services (1 mentions)
Osmium tetroxide, by Science Services (1 mentions)
Embed 812 kit, by Electron Microscopy Sciences (1 mentions)
Em uc6 ultramicrotome, by Leica (1 mentions)
Azur 2, by Merck Group (1 mentions)
Methylene blue, by Merck Group (1 mentions)
Diamond knife, by Leica (1 mentions)
Em uc6, by Leica (1 mentions)
Glutaraldehyde, by Science Services (1 mentions)
Osmium tetroxide, by Serva Electrophoresis (1 mentions)
Epoxide resin, by Serva Electrophoresis (1 mentions)
Thincert cell culture insert, by Greiner (1 mentions)
9 protocols using uranyl acetate
1
Transmission Electron Microscopy Sample Preparation
2
Transmission Electron Microscopy Tissue Preparation
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Organs were fixed in 2.5% glutaraldehyde/4% formaldehyde in Tris-HCl (10 mM, pH = 7.4, VWR, Austria) and stored at 4°C until further treatment. Samples were postfixed for 20 minutes at room temperature with 1% osmium tetroxide in water (Science Services, Germany). Dehydration in a graded ethanol series (0-30-50-70-90-95-100%, VWR, Austria) was followed by a stepwise embedding in epoxy resin (EMbed-812 Kit, Electron Microscopy Sciences, USA). Polymerization was performed at 60°C for 2 days in beem capsules (easy-molds, Electron Microscopy Sciences, USA). Semithin sections were cut with an EM UC6 ultramicrotome (Leica Microsystems, Vienna, Austria) and stained with 0.5% Azur II and 1% methylene blue in 1% sodium borate (Sigma-Aldrich, Austria). Ultrathin sections were transferred to copper grids (Science Services, Germany) and stained for 15 minutes in uranyl acetate (Science Services, Germany) and for 5 minutes in lead citrate (Science Services, Germany), each at room temperature. Grids were analyzed with a Tecnai twin G20 transmission electron microscope (FEI Company, Eindhoven, The Netherlands) equipped with a LaB6 cathode and operated at 120 kV.
3
Resin Embedding and Sectioning Protocol
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After freeze substitution, samples were washed three times in pure acetone and infiltrated with resin as previously published [31 (link)]. In brief, samples were gradually infiltrated with Epon/Araldite resin (one part resin: three parts acetone) for 1 hour; 1: 1 for 2 hours; 3: 1 for 2 hours, and 100% resin for 1 hour, then 100% resin overnight, then 100% resin for 1 hour and thin-layer embedded [31 (link)]. Resin-infiltrated samples were polymerized for three days at 60°C.
Selected samples were re-mounted on dummy blocks for ultramicrotomy [31 (link)]. Serial thin (70 nm) sections for routine transmission electron microscopy and semi-thick (300 nm) sections for electron tomography were cut using an ultramicrotome (EM UC6, Leica Microsystems, Austria) equipped with a diamond knife (Diatome, Switzerland). Sections were collected on Formvar-coated copper slot grids and post-stained with 2% (w/v) uranyl acetate (Science Services, USA) in 70% methanol for 10 min, followed by 0.4% (w/v) lead citrate (Science Services, USA) in double-distilled water for 5 min. In addition, colloidal gold (20 nm diameter, BBI, UK) was attached to the semi-thick sections to serve as fiducial markers for the calculation of electron tomograms.
Selected samples were re-mounted on dummy blocks for ultramicrotomy [31 (link)]. Serial thin (70 nm) sections for routine transmission electron microscopy and semi-thick (300 nm) sections for electron tomography were cut using an ultramicrotome (EM UC6, Leica Microsystems, Austria) equipped with a diamond knife (Diatome, Switzerland). Sections were collected on Formvar-coated copper slot grids and post-stained with 2% (w/v) uranyl acetate (Science Services, USA) in 70% methanol for 10 min, followed by 0.4% (w/v) lead citrate (Science Services, USA) in double-distilled water for 5 min. In addition, colloidal gold (20 nm diameter, BBI, UK) was attached to the semi-thick sections to serve as fiducial markers for the calculation of electron tomograms.
4
Ultrastructural Analysis of Microglia
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To analyse the ultrastructure of triple labelled microglia, these cells were sorted on a permeable support PET membrane with 1um size pores (ThinCert™ Cell Culture Inserts, Greiner Bio-One) and prepared for electron microscopy. Fixation was performed in 2.5% (vol/vol) glutaraldehyde (Science Services, Munich, Germany) in 0.2 M sodium cacodylate buffer (Merck, Darmstadt, Germany) at 4 °C overnight. Samples were post-fixed in osmium tetroxide (1%, vol/vol, Serva, Heidelberg, Germany) and stained in 2% uranyl acetate (Science Services, Munich, Germany) in 70% ethanol. After dehydration, membranes were embedded in epoxide resin (Araldite, Serva, Heidelberg, Germany). Finally, blocs were used for ultramicrotomy and sections were stained in 0.4% (vol/vol) lead citrate (Merck, Darmstadt, Germany). Samples were analysed using an EM10 electron microscope (Carl Zeiss, Oberkochen, Germany).
5
Ultrastructural Analysis of CFSC-2G Cells
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Electron microscopic analysis of CFSC-2G cells was essentially conducted as described before [6 (link)]. In brief, cells were fixed in 1× phosphate buffered saline (PBS) containing 3% glutaraldehyde, washed in 0.1 M Soerensen’s phosphate buffer (Merck, Darmstadt, Germany), and fixed in a solution of 1% osmium tetroxide (OsO4) (Roth, Karlsruhe, Germany) solved in 25 mM sucrose buffer (Merck). Fixed cells were dehydrated and subsequently incubated in propylene oxide (Serva, Heidelberg, Germany) in a mixture of Epon resin (Serva) and propylene oxide (1:1) and pure Epon. Finally, ultrathin sections (70–100 nm) were prepared and packed upon Cu/Rh grids (HR23 Maxtaform, Plano GmbH, Wetzlar, Germany). Contrast was enhanced by staining with 0.5% uranyl acetate and 1% lead citrate (both Science Services, Munich, Germany). The samples were analyzed at an acceleration voltage of 60 kV using a Zeiss Leo 906 (Carl Zeiss AG, Oberkochen, Germany) transmission electron microscope and depicted images were taken at magnifications of 2156× to 35,970×.
6
Micellar Dispersion Imaging by TEM
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For TEM analysis, micellar dispersions (30 mg/mL) were diluted 200 times with Milli-Q water. The samples were left to adsorb onto 200-mesh glow discharged formvar-carboncoated nickel grids (Maxtaform) for 10 min, followed by 3 rounds of washing with Milli-Q water. Negative staining of the samples was carried out with 0.5% uranyl acetate (Science Services GmbH). Samples were imaged using a LEO 906 (Carl Zeiss) microscope at an acceleration voltage of 60 kV.
7
Ultrastructural Imaging of Spinal Cord
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The animals used for the transmission electron microscopy (EM) experiment were perfused directly after the experiment with 5 ml of HBSS, followed by 30 ml of fixative (2.5% glutaraldehyde, 4% PFA in phosphate buffer; 0.1 M). The spinal cord at the laminectomy site was immersed in the same solution during the perfusion. After overnight fixation, the spinal cord was extracted and further post-fixed overnight at 4°C in the same fixative. The area below the laminectomy was collected and post-fixed with 2% OsO4 and 1.5% ferricyanide (Science Services), dehydrated by ethanol, then acetone and finally Epon-embedded (Serva). Then 50-nm ultrathin sections from the area corresponding to the imaging site in the two-photon acute imaging experiment were contrasted with 4% uranyl acetate (Science Services) and lead citrate (Sigma). The imaging was done on a transmission electron microscope JEM 1400 plus (JEOL) equipped with an 8 megapixel camera (Ruby, JEOL). Large areas covering the first 20–30 µm from the dorsal pial surface of the spinal cord were imaged using the Shuttle-and-Find software at ×8000. Images were processed using the open-source image analysis software, Fiji.
8
Optimized Electron Microscopy Staining
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Samples were processed, by submerging slides in buffers, fixatives, solvents, and resins, in standard staining cuvettes (Fig. 3b ). The customized rOTO-protocol, was based on (Willingham & Rutherford, 1984 (link)), with 1% OsO4 (Science Services, Munich, Germany) and 1% K4Fe(CN)6 (Merck) in cacodylate buffer for 30 min, washed three times in aqua bidest., incubated with 1% thiocarbohydrazide (Merck) in aqua bidest. for 30 min, washed with aqua bidest. three times, followed by post-fixation with 1% OsO4 in aqua bidest. for 30 min. The samples were rinsed three times with aqua bidest. and dehydrated in a graded series of acetone (10, 20, 40, 60, 80, 100%), with a 1% uranyl acetate (Science Services) step in 20% acetone for 30 min.
9
Ultrastructural Analysis of miR-200c Zebrafish
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Followed by miR-200c injection, zebrafish larvae were fixed in solution D overnight, washed 3x in 0.1 M cacodylate buffer and postfixed in 1% OsO4 for 1 h. Tissues were once again washed, dehydrated and embedded in EPON (recipe/protocol from EMS, Hatfield, PA, USA). Semi-thin (300 nm) and ultra-thin (90 nm) sections were cut with a Leica UC-6 Microtome (Leica Microsystems GmbH, Wetzlar, Germany). Sections obtained were mounted onto formvar-coated Ni slot grids (EMS, Electron Microscopy Sciences, Hatfield, PA, USA). Grids were stained for 30 min in 5% uranyl acetate (Science Services, München, Germany), followed by 0.1% lead citrate (Science Services, München, Germany) for 15 min.
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