Cell culture preparations were fixed in 2.5% glutaraldehyde phosphate buffer overnight at 4°C, washed with PBS and postfixed with 1% osmium tetroxide for 2 h at 4°C. After dehydration in a graded ethanol series, the cells were embedded in Epoxy EMbed-812 resin (Electron Microscopy Sciences, Hatfield, PA, USA), followed by polymerizing for 48 h at 60. Ultrathin sections were stained with lead citrate and uranyl acetate. The sections were then examined and photographed with a transmission electron microscope (H-7650; Hitachi Ltd., Tokyo, Japan).
Epoxy embed 812 resin
Manufactured by Electron Microscopy Sciences
Sourced in United States
Epoxy EMbed-812 resin is a low-viscosity epoxy resin formulated for embedding and sectioning of biological and material science samples for electron microscopy. It has a low viscosity and polymerizes at moderate temperatures to provide a hard, durable embedding medium.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer z1, by Zeiss (9 mentions)
Ultracut e, by Leica (3 mentions)
Em uc7 ultramicrotome, by Leica (2 mentions)
H 7650, by Hitachi (1 mentions)
Ultracut e ultramicrotome, by Leica (1 mentions)
Campesterol, by Merck Group (1 mentions)
Polyvinylpolypyrrolidone, by Merck Group (1 mentions)
Potassium hydroxide solution, by Merck Group (1 mentions)
Uranyl acetate, by Merck Group (1 mentions)
Hplc grade water, by Merck Group (1 mentions)
Osmium tetroxide, by Merck Group (1 mentions)
Sodium acetate ch3coona, by Merck Group (1 mentions)
Triton x 114, by Merck Group (1 mentions)
Sodium phosphate dibasic na2hpo4, by Merck Group (1 mentions)
Stigmasterol, by Merck Group (1 mentions)
β mercaptoethanol β me, by Merck Group (1 mentions)
β sitosterol, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Gallic acid, by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
9 protocols using epoxy embed 812 resin
1
Cell Fixation and Embedding for TEM
2
Ultrastructural Synaptic Analysis in Mice
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Animals were euthanized and their eyes enucleated and fixed overnight in freshly prepared 2% (w/v) paraformaldehyde and 2% (w/v) glutaraldehyde in sodium cacodylate buffer (0.1 M, pH 7.4). After dissection of the cornea and lens, optic cups were post-fixed for 1 h with 1% (w/v) OsO4 and 1% (w/v) K3Fe(CN)6 in ultrapure water, dehydrated through a graded series of ethanol solutions and embedded in Epoxy EMbed-812 resin (EMS, Electron Microscopy Sciences). Semi-thin (0.5 μm) and ultra-thin sections were obtained using an Ultracut E ultramicrotome (Leica). Semi-thin sections were stained with Toluidine Blue and mounted with Entellan and images were obtained using a light microscope (Axio Observer Z1, Zeiss) with 20× and 100× oil immersion objectives. Ultra-thin sections were contrasted with uranyl acetate and lead citrate, and analyzed at the NUCLEUS electron microscopy facility at the University of Salamanca using a Tecnai Spirit Twin 120 kv electron microscope with a CCD Gatan Orius SC200D camera with DigitalMicrograph™ software. The proportions of the different types of synapses were quantified in ultra-thin sections from 3 mice of each genotype by analyzing between 58 and 86 synapses per mouse.
3
Electron Microscopy Sample Preparation
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Cell culture preparations were fixed in 2% formaldehyde and 2% glutaraldehyde in phosphate buffer for 30 min at 4 °C. Samples were then post-fixed with 1% osmium tetroxide in water, dehydrated through a graded ethanol series, and embedded in Epoxy EMbed-812 resin (Electron Microscopy Sciences). Ultrathin sections were obtained with a Leica EM UC7 ultramicrotome, contrasted with uranyl acetate and lead citrate, and analysed using a Tecnai Spirit Twin 120 kV electron microscope with a CCD Gatan Orius SC200D camera with DigitalMicrograph™ software. Procedures were performed at the Electron Microscopy Facilities-NUCLEUS of the University of Salamanca.
4
Comprehensive Analysis of Bioactive Compounds
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Acetonitrile, ethyl acetate, methanol (MeOH), hexane, water HPLC grade (H2O), β-mercaptoethanol (β-ME), 2,2-diphenyl-1-picrylhydrazyl (DPPH), boric acid (H3BO3), hydrochloric acid (HCl), glutaraldehyde, osmium tetroxide, polyvinylpolypyrrolidone (PVPP), potassium hydroxide solution (KOH 0.1 M), sodium acetate (CH3COONa), sodium phosphate monobasic (NaH2PO4), sodium phosphate dibasic (Na2HPO4), Triton X-114 and uranyl acetate were acquired from Sigma-Aldrich (St. Louis, MO, USA) along with 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), linoleic acid, β-sitosterol, stigmasterol, campesterol, catechin, gallic acid and vanillin. Epoxy EMbed 812 resin was purchased from Electron Microscopy Sciences (Hatfield, PA, USA). Acetic acid (CH3COOH), ethanol (EtOH), sodium carbonate (Na2CO3) and KOH were purchased from DEQ (Monterrey, Nuevo León, Mexico).
5
Ultrastructural analysis of Hf-RPE cells
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Hf-RPE cells were fixed with 2% PFA and 2% glutaraldehyde in 0,1 M cacodylate buffer, pH 7,4, for 24 h at 4°C, and post-fixed during 2 h in darkness with 1% OsO4 (v/v) and 1% K3Fe(CN)6 (v/v) diluted in ultrapure water. Then, cells were washed with distilled water and dehydrated using a graded ethanol series, and a final step in propylene oxide prior to resin infiltration. Following on, samples were embedded in Epoxy EMbed-812 resin (Electron Microscopy Sciences), ultrathin sections were obtained using an ultramicrotome Ultracut E (Leica), which were contrasted with uranyl acetate and lead citrate and analyzed using a JEOL JEM-1011 HR electron microscope with a CCD Gatan ES1000W camera with iTEM software at the Electron Microscopy Facilities-NUCLEUS of the University of Salamanca. Minor contrast and brightness adjustments were performed with Adobe Photoshop CS5 Extended software and Leica Confocal software. Height and length measurements were carried out with ImageJ software.
6
Transmission Electron Microscopy Workflow
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Transmission electron microscopy was performed as previously reported [21 (link)]. Cell culture preparations were fixed in 2% formaldehyde and 2% glutaraldehyde in phosphate buffer for 30 min at 4 °C. Samples were then post-fixed with 1% osmium tetroxide in water, dehydrated through a graded ethanol series, and embedded in Epoxy EMbed-812 resin (Electron Microscopy Sciences, Hatfield, PA, USA). Ultrathin sections were obtained with a Leica EM UC7 ultramicrotome, contrasted with uranyl acetate and lead citrate, and analyzed using a Tecnai Spirit Twin 120 kV electron microscope with a CCD Gatan Orius SC200D camera with DigitalMicrograph™ software. Procedures were performed at the Electron Microscopy Facilities-NUCLEUS of the University of Salamanca.
7
Ultrastructural Analysis of Leaf Cells
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Three plantlets at the three fully-developed leaves stage (Zadoks stage 13;
[114 (link)]) from the glasshouse experiment, irrigated at field capacity, were collected from each genotype. Small segments (1 mm) from the central part of the third-leaf blades were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) at 4°C. After three washes in 0.1 M phosphate buffer (pH 7.4) the samples were incubated in 1% osmium tetroxide and 2.5% potassium ferrocyanide for 2 h at 4°C. After ten washes for 10 min each with 0.1 M sodium acetate, and incubation for 30 min in 0.5% uranyl acetate followed by two washes for 10 min in 0.1 M sodium acetate at 4°C, the samples were dehydrated in increasing concentrations of ethanol followed by two washes with sodium acetate and finally embedded in epoxy EMBED-812 resin (Electron Microscopy Sciences) for three days at 60°C. Ultrathin sections (70 nm) contrasted with Reynolds’ lead citrate stain were examined using a Zeiss TEM 910 transmission electron microscope.
[114 (link)]) from the glasshouse experiment, irrigated at field capacity, were collected from each genotype. Small segments (1 mm) from the central part of the third-leaf blades were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) at 4°C. After three washes in 0.1 M phosphate buffer (pH 7.4) the samples were incubated in 1% osmium tetroxide and 2.5% potassium ferrocyanide for 2 h at 4°C. After ten washes for 10 min each with 0.1 M sodium acetate, and incubation for 30 min in 0.5% uranyl acetate followed by two washes for 10 min in 0.1 M sodium acetate at 4°C, the samples were dehydrated in increasing concentrations of ethanol followed by two washes with sodium acetate and finally embedded in epoxy EMBED-812 resin (Electron Microscopy Sciences) for three days at 60°C. Ultrathin sections (70 nm) contrasted with Reynolds’ lead citrate stain were examined using a Zeiss TEM 910 transmission electron microscope.
8
Ultrastructural Analysis of Mouse Retinal Synapses
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Animals were euthanized and their eyes enucleated and xed overnight in freshly prepared 2% paraformaldehyde and 2% glutaraldehyde in sodium cacodylate buffer (0.1 M, pH 7.4). After dissection of the cornea and lens, optic cups were post-xed for 1 hour with 1% OsO 4 (v/v) and 1% K 3 Fe(CN) 6 (v/v) in ultrapure water, dehydrated through a graded series of ethanol solutions and embedded in Epoxy EMbed-812 resin (EMS, Electron Microscopy Sciences). Semi-thin (0.5 µm) and ultra-thin sections were obtained using an Ultracut E ultramicrotome (Leica). Semi-thin sections were stained with Toluidine Blue and mounted with Entellan and images were obtained using a light microscope (Axio Observer Z1, Zeiss) with 20 × and 100 × oil immersion objectives. Ultra-thin sections were contrasted with uranyl acetate and lead citrate, and analyzed at the NUCLEUS electron microscopy facility at the University of Salamanca using a Tecnai Spirit Twin 120 kv electron microscope with a CCD Gatan Orius SC200D camera with DigitalMicrograph™ software. The proportions of the different types of synapses were quanti ed in ultrathin sections from 3 mice of each genotype by analyzing over 60 synapses per mouse.
9
Ultrastructural Analysis of Mouse Retinal Synapses
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Animals were euthanized and their eyes enucleated and fixed overnight in freshly prepared 2% paraformaldehyde and 2% glutaraldehyde in sodium cacodylate buffer (0.1 M, pH 7.4). After dissection of the cornea and lens, optic cups were post-fixed for 1 hour with 1% OsO4 (v/v) and 1% K3Fe(CN)6 (v/v) in ultrapure water, dehydrated through a graded series of ethanol solutions and embedded in Epoxy EMbed-812 resin (EMS, Electron Microscopy Sciences). Semi-thin (0.5 m) and ultra-thin sections were obtained using an Ultracut E ultramicrotome (Leica). Semi-thin sections were stained with Toluidine Blue and mounted with Entellan and images were obtained using a light microscope (Axio Observer Z1, Zeiss) with 20× and 100× oil immersion objectives.
Ultra-thin sections were contrasted with uranyl acetate and lead citrate, and analyzed at the NUCLEUS electron microscopy facility at the University of Salamanca using a Tecnai Spirit Twin 120 kv electron microscope with a CCD Gatan Orius SC200D camera with DigitalMicrograph™ software. The proportions of the different types of synapses were quantified in ultra-thin sections from 3 mice of each genotype by analyzing over 60 synapses per mouse.
Ultra-thin sections were contrasted with uranyl acetate and lead citrate, and analyzed at the NUCLEUS electron microscopy facility at the University of Salamanca using a Tecnai Spirit Twin 120 kv electron microscope with a CCD Gatan Orius SC200D camera with DigitalMicrograph™ software. The proportions of the different types of synapses were quantified in ultra-thin sections from 3 mice of each genotype by analyzing over 60 synapses per mouse.
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