Samples were fixed in 50 mM phosphate buffer containing 2.5% (v/v) glutaraldehyde and 4% formaldehyde (v/v) for 3.5 hours then washed twice with 100 mM phosphate buffer. A secondary fix of 1% osmium tetroxide in 50 mM phosphate buffer was carried out for 40 minutes then the preparations were washed twice with 50 mM phosphate buffer. Samples were dehydrated then infiltrated and fixed in Spurrs resin (Agar Scientific) (25, 50, 75 and 100%) with overnight polymerization at 70°C. Sections were stained with saturated uranyl acetate and Reynolds lead citrate and viewed using a Tecnai 12 Bio Twin TEM operating at 120 kV. The chemical composition of nanoparticles was confirmed using Energy-dispersive X-ray spectroscopy (EDX) with an Oxford INCA analysis system.
Spurrs resin
Manufactured by Agar Scientific
Sourced in United Kingdom
Spurrs resin is a low-viscosity epoxy resin formulated for embedding and infiltrating biological specimens for electron microscopy. It is designed to provide a firm, durable support for sections during cutting and examination.
Automatically generated - may contain errors
Lab products found in correlation
Ultracut s, by Leica (2 mentions)
Osmium tetroxide, by Agar Scientific (1 mentions)
Glutaraldehyde 3, by Agar Scientific (1 mentions)
1010 microscope, by JEOL (1 mentions)
Ultracut r ultramicrotome, by Leica (1 mentions)
Jem 1230, by JEOL (1 mentions)
Ultra cut microtome, by Agar Scientific (1 mentions)
Tecnai t12 transmission electron microscope tem, by Thermo Fisher Scientific (1 mentions)
Fei tecnai t12, by Thermo Fisher Scientific (1 mentions)
8 protocols using spurrs resin
1
Transmission Electron Microscopy of Nanoparticles
2
Transmission Electron Microscopy Sample Preparation
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The samples were fixed by glutaraldehyde 3% (Agar Scientific, UK) in phosphate buffer at pH:7.2 overnight. The samples were then kept in Osmium tetroxide 1% (Agar Scientific, UK) in phosphate buffer pH:7.2 for 1 h. After each step, the samples were washed by 0.1 M phosphate buffer pH:7.2. Next, samples were dehydrated bycethanol and acetone. The samples were then resin infiltrated by spurr’s resin (Agar Scientific), acetone and pure spur. They were polymerized in 70 °C oven and thin sections (80 nm thickness) were prepared using RMC MT-7000 ultramicrotome. The samples were then stained with uranyl acetate and lead citrate and were examined at 50 kV by Zeiss EM900 transmission electron microscope.
3
Plant Tissue Ultrastructural Imaging
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Plant leaf tissues were fixed in 2.5% (v/v) glutaraldehyde, 4% formaldehyde (v/v) in 50 mM phosphate buffer for 3.5 hours a secondary fix of 1% osmium tetroxide. Samples were then dehydrated through a 25–100% acetone series and infiltrated with Spurrs resin (Agar Scientific) (25, 50 and 75%) with overnight polymerisation at 343 K. Sections were mounted on 400 mesh thin-bar Athene grids (Agar Scientific), stained with saturated uranyl acetate and Reynolds lead citrate and viewed using a Tecnai 12 Bio Twin TEM operating at 120 kV.
4
Ultrastructural Analysis of Apoptotic β-cells
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Isolated islets were fixed in 4% paraformaldehyde and 0.5% glutaraldehyde in phosphate buffer for 1 h, post-fixed in 1% osmium tetroxide, block stained in 2% uranyl actetate, dehydrated in graded ethanol and embedded in Spurr’s resin (Agar Scientific, Stansted, UK). Ultrathin sections (70 nm) were cut onto Ni2+ grids, contrasted with 2% uranyl acetate and lead citrate, and examined in a Jeol 1010 microscope (Welwyn Garden City, UK) with an accelerating voltage of 80 kV.
β-Cell apoptosis was assessed from electron micrographs as the number of β-cells with apoptotic nuclei (that is, displaying typical chromatin condensation): β-cells (135–150 from n=3–9 mice; n=3 islets per mouse) were identified by the presence of characteristic insulin granules.
β-Cell apoptosis was assessed from electron micrographs as the number of β-cells with apoptotic nuclei (that is, displaying typical chromatin condensation): β-cells (135–150 from n=3–9 mice; n=3 islets per mouse) were identified by the presence of characteristic insulin granules.
5
TEM Analysis of Plant Leaf Ultrastructure
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For transmission electron microscopy, 2-mm2 pieces from the middle sections of the youngest and fully expanded leaves were dissected and immediately fixed in 2.5 % (v/v) glutaraldehyde in 0.1 M phosphate buffer (pH 7.0) overnight at 4 °C. The samples were then washed three times with the same buffer and post-fixed in 1 % osmium tetroxide overnight at 4 °C. After being washed in the same buffer, the leaf tissues were passed through an ethanol dehydration series, and then infiltrated and embedded in Spurr’s resin (Agar Scientific, Essex, UK). Sections were cut using an Ultracut R ultramicrotome (Leica, Wetzlar, Germany). The thin sections were stained with 2 % uranyl acetate and lead citrate [55 (link)], and then observed and photographed under a transmission electron microscope (JEM-1230, JEOL Ltd, Tokyo, Japan). For each treatment, three leaf samples were examined, and approximately 130 mesophyll cells were randomly chosen for the observations.
6
Transmission Electron Microscopy Specimen Preparation
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Cells grown on polyester filters were immersion fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 10 minutes at room temperature and prepared for electron microscopy (EM) by standard methods. Briefly, filters were stained with osmium tetroxide (1% v/v in 0.1 M phosphate buffer) and uranyl acetate (2% w/v in distilled water), dehydrated through increasing concentrations of ethanol (70%–100%) and acetone and embedded in Spurr's resin (Agar scientific, Reading, U.K.). Ultrathin sections (50–80 nm) were prepared by use of a Reichart‐Jung ultracut microtome and mounted on nickel grids (Agar Scientific, Stanstead, Essex, U.K.). Sections were counterstained with lead citrate and uranyl acetate and examined on a JOEL 1010 transmission electron microscope (JOEL USA, Peabody, MA).
7
Ultrastructural Analysis of Egg Capsules
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Three pieces (~ 1 cm2) of each of the egg capsules were dissected and fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer as per the scanning electron microscopy samples. Egg capsule pieces were then rinsed in 0.1 M phosphate buffer and fixed in 1% osmium tetroxide (OsO4) with 1% potassium ferrocyanide in 0.1 M phosphate buffer at room temperature for 1 h. Samples were rinsed in 0.1 M phosphate buffer and then dehydrated in a series of ethanol. Ethanol was gradually replaced by Spurr’s resin (Agar Scientific, Essex, UK) in 25% increments. Egg capsule pieces were then transferred to individual BEEM capsules and polymerised at 60 °C overnight. Ultrathin sections (70 nm) were cut using a Ultracut S (Leica, Wetzlar, Germany) microtome with glass knives and placed on 200 mesh copper grids. Three to five grids were made per resin block. Grids were post stained with 2% uranyl acetate for 10 min, rinsed three times in deionised warm water and then stained with Reynold’s lead citrate stain surrounded by sodium hydroxide pellets for 10 min. Grids were rinsed three times with deionised warm water and then air dried. Sections were viewed and imaged with a FEI Tecnai T12 Transmission Electron Microscope (TEM) at 80 kV.
8
Ultrastructural Analysis of Uterine and Placental Tissues
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Uterine and placental tissues were cut into approximately 1 mm 3 portions and fixed in 2.5 % glutaraldehyde in 0.1 M phosphate buffer for 1 h. Tissue was then rinsed in 0.1 M phosphate buffer and fixed in 1% osmium tetroxide (OsO4) in 0.1 M phosphate buffer at room temperature for 1 h. Samples were rinsed in 0.1 M phosphate buffer and then dehydrated in a series of ethanol. Ethanol was gradually replaced by Spurrs resin (Agar Scientific, Essex, UK) in 25% increments. Each piece of tissue was embedded in BEEM capsules and polymerised at 60 ˚C overnight. Resin blocks were removed from their capsules and ultrathin sections of approximately 70 nm were cut using a Ultracut S (Leica, Wetzlar, Germany) microtome with glass knives and placed on 200 mesh copper grids. At least three grids were made per resin block. Grids were post-stained with 2% uranyl acetate for 10 min and then rinsed with warm water. Grids were then post-stained with Reynold's lead citrate stain surrounded by sodium hydroxide pellets for 10 min and then rinsed in warm water. Grids were allowed to air dry and then sections were imaged with a FEI Tecnai T12 (FEI, USA) at 120 kV. The contrast was adjusted, and labels were added to all images using Adobe photoshop 2021 (22.1.1).
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