The plant samples were fixed first in fixation buffer I (5% glutaraldehyde and 0.1 M phosphate, pH 7.4) for 4 h at room temperature and then in fixation buffer II (2% osmium tetroxide and 0.1 M phosphate, pH 7.4) at 4 °C overnight. After one wash in phosphate buffer and two washes in double-distilled water, the samples were stained for 1 h in 1% (m/v) uranyl acetate. After another wash in double-distilled water, the samples were dehydrated through a gradient of alcohol (from 30%, 50%, 70%, to 85%) and then embedded in Spurr’s resin (Sigma-Aldrich). Ultrathin sections of 70 nm were prepared using the UC7 μLtramicrotome (Leica Microsystem) and mounted on the AG100M molybdenum grids with a single slot (Agar Scientific) to minimize the background for copper. The sections were stained with uranyl acetate and lead citrate, observed, and photographed using a Tecnai G2 20 Twin electron microscope (FEI) at 120 kV. EF-TEM for copper element analysis was carried out on the same specimen using a JEM-2100F field-emission high-resolution transmission electron microscope equipped with the energy filter (JEOL). Three individual leaves were used for each genotype and at least 10 cells were analyzed per sample.
Spurr s resin
Manufactured by Merck Group
Sourced in United States
Spurr's resin is a low-viscosity epoxy resin commonly used in electron microscopy sample preparation. It is designed to provide a stable embedding matrix for the preservation and support of delicate biological specimens during the sectioning process. The resin's low viscosity allows for effective infiltration and embedding of the sample, ensuring optimal structural integrity during subsequent imaging and analysis.
Automatically generated - may contain errors
Lab products found in correlation
H 7500, by Hitachi (3 mentions)
Toluidine blue o, by Merck Group (2 mentions)
Lrw resin, by Polysciences (2 mentions)
M109 turbo, by Zeiss (2 mentions)
Ultramicrotome, by Reichert Technologies (2 mentions)
Tecnai g2 20 twin electron microscope, by Thermo Fisher Scientific (1 mentions)
Jem 2100f field emission high resolution transmission electron microscope, by JEOL (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Rnase a, by Merck Group (1 mentions)
Glycerol gelatin, by Merck Group (1 mentions)
Hyperfilm mp, by Cytiva (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Oligotex, by Qiagen (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Uct ultramicrotome, by Zeiss (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Ultra 45 diamond knife, by Electron Microscopy Sciences (1 mentions)
Uc7 ultramicrotome, by Electron Microscopy Sciences (1 mentions)
16 protocols using spurr s resin
1
Ultrastructural Analysis of Plant Leaves
2
Digoxygenin-Labeled dT50 Probe Preparation
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Digoxygenin-11-uridine-5′-triphosphate (Dig-11-dUTP), Digoxygenin-specific mouse antibodies and antibody blocking reagent (Boehringer Mannheim), fluorescein (FITC)-conjugated goat anti-mouse antibodies (Cappel, Organon Teknica), zymolyase (ICN), terminal transferase (USB), RNAse A (Sigma), RNAse T1 (Boehringer Mannheim), Oligotex (Qiagen), formaldehyde (Fluka), paraformaldehyde (Sigma), EM grade glutaraldehyde (VWR), Spurr’s resin (Sigma Aldrich), glycerol-gelatin (Sigma), b-mercaptoethanol (Sigma), vanadyl ribonucleoside complex (Gibco BRL), p-phenylenedamine (Sigma), 4,6-diamidino-2-phenylindole (DAPI, Sigma), poly-L-lysine (Sigma), 16 well NUNC chamber slides (American Scientific), Hyperfilm-mp (Amersham).
dT50 Probe preparation: oligo dT50mers were end labeled with Dig-11-dUTP using terminal transferase as described in the product information. 40 ul of Digoxygenin-labeled dT50 probe (approximately 0.5 pmoles/ml) was added to 500 ul 2x hybridization mix (10x SSC, 1% Denhardts, 0.02% tween, 20 mM vanadyl complex in DEPC-treated gdH2O), 140 ul DEPC-treated gdH2O and 300 ul formamide to yield the working probe mix. Negative control probes consisting of random oligo 50mers were end labeled with digoxygenin in a similar manner and used to verify probe specificity in the in situ hybridization assay.
dT50 Probe preparation: oligo dT50mers were end labeled with Dig-11-dUTP using terminal transferase as described in the product information. 40 ul of Digoxygenin-labeled dT50 probe (approximately 0.5 pmoles/ml) was added to 500 ul 2x hybridization mix (10x SSC, 1% Denhardts, 0.02% tween, 20 mM vanadyl complex in DEPC-treated gdH2O), 140 ul DEPC-treated gdH2O and 300 ul formamide to yield the working probe mix. Negative control probes consisting of random oligo 50mers were end labeled with digoxygenin in a similar manner and used to verify probe specificity in the in situ hybridization assay.
3
Anther Structure Analysis by Microscopy
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For anther wall structure analysis with confocal microscopy, the anthers of precisely measured sizes were dissected and stored in 70% ethanol for fixation. Fixed anthers were then stained by propidium iodide and visualized on a Leica SP8 confocal microscope20 (link). Meiocytes were extruded and stained by DAPI (4′,6-diamindino-2-phenylindole)33 (link). The Alexander′s staining solution34 was used to test the viability of the pollen grains in the permissive conditions. For TEM35 , anthers were dissected and fixed in fresh 0.1 M PIPES buffer (pH 6.8). A 2% osmium tetroxide stain was applied with brief washes, followed by dehydration in an acetone gradient. Specimens were infiltrated using a gradient of Spurr’s resin (Sigma-Aldrich) and embedded. The sections were cut using a Leica UCT ultramicrotome, stained in urayl and lead salts, and observed using a Zeiss TEM instrument with a LEO 912 AB energy filter. Semi-thin sections were obtained with the same method as TEM except for the heavy metal staining steps, and imaged with Aperio Digital Pathology Slide Scanner with a 40× lens.
4
Ultrastructural Analysis of Nerve Grafts
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From the selected nerve samples, a 1-mm-long segment from the middle of the graft, or the corresponding site of naive control sciatic nerve, was dissected for transmission electron microscopy as previously described (Zhan et al., 2013). After fixation with 2.5% glutaraldehyde/2% paraformaldehyde for 24 hours, the samples were post-fixed in 1% OsO4 for 2 hours at 4 °C and dehydrated in a graded acetone series. The samples were then embedded with Spurr's resin (Sigma) for ultrathin transverse sectioning (70 nm) and counterstaining with 2% uranyl acetate and lead citrate. The stained ultrathin sections were observed with a transmission electron microscope (H-7500, Hitachi). Six random non-overlapping images of each sample were captured for the following assessments: (1) myelin thickness; (2) axonal diameter; (3) G-ratio of the myelinated nerve fiber, which was determined by dividing axon diameter by the diameter of the whole myelinated fiber; and (4) myelinated axon ratio, which was calculated by counting the number of myelinated axons divided by the number of total axons. The measurements were obtained using Image-Pro Plus software (Media Cybernetics, Bethesda, MD, USA).
5
Ultrastructural Analysis of 3D HIEs
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HIEs (3D) embedded in Matrigel were fixed in a solution of 2% paraformaldehyde + 2.5% glutaraldehyde + 2 mmol/L calcium chloride in 0.1 mol/L cacodylate buffer (pH = 7.4) for 5–7 days at 4°C. They were postfixed in 1% osmium tetroxide in 0.1 mol/L cacodylate buffer for 1 hour and stained en bloc with saturated aqueous uranyl acetate. After a routine dehydration sequence, tissue pieces gradually were infiltrated in a gradient series of Spurr’s Low Viscosity resin (Sigma-Aldrich) and ethanol, then embedded in fresh Spurr’s resin and polymerized at 60°C for 3 days. Thin sections (55–60 nm) were cut on a Leica (Wezlar, BRD) UC7 ultra-microtome using a Diatome Ultra 45 diamond knife. Sections were viewed on a Hitachi (Tokyo, Japan) H7500 transmission electron microscope set to 80 kV. Images were collected using an AMT XR-16 digital camera and AMT Image Capture (Advanced Microscopy Techniques Corp, Woburn, MA), v602.600.51 software.
6
Cytotoxicity Evaluation of Nanomaterials
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The antibiotic G418 and the culture medium Dulbecco′s Modified Eagle′s Medium (DMEM) were from Sigma-Aldrich (St. Louis, MO, USA). The CellTiter 96®Aqueous One Solution Cell Proliferation Assay kit was from Promega (Tecnolab, Buenos Aires, Argentina). Ferric chloride hexahydrate, sodium dodecyl sulfate (SDS), and hematoxylin and eosin (H&E) dyes were provided by Biopack (Buenos Aires, Argentina). Ferrous sulfate heptahydrate and potassium ferrocyanide were from Mallinckrodt Chemical Works (Saint Louis, MO, USA). Sodium hydroxide and acetic acid were purchased from Cicarelli (San Lorenzo, Santa Fe, Argentina). Neutral red (3-amino-7-dimethylamino-2-methyl-phenazine hydrochloride), 2′,7′-dichlorofluorescin diacetate (DCFDA), osmium tetroxide, uranyl acetate, lead citrate, and Spurr’s resin were acquired by Sigma (Sigma-Aldrich, Saint Louis, MO, USA). All other reagents were of analytical grade.
7
Ultrastructure Analysis of Sciatic Nerve
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Sciatic nerve explants cultured for 5 or 8 div (n = 3 per group) were selected for assessment using transmission electron microscopy as previously described (Pan et al., 2017; Wen et al., 2017). Briefly, the sciatic nerve explants were immersed in 2.5% glutaraldehyde for 24 hours, postfixed in 1% osmium tetroxide for 2 hours at 4°C, dehydrated in a graded acetone series, and embedded in Spurr’s resin (Sigma-Aldrich). After ultrathin sectioning (70 nm), the sections were stained with 2% uranyl acetate and lead citrate, and images were captured under a transmission electron microscope (H-7500; Hitachi, Tokyo, Japan). Based on the protocol described by Jang et al. (2016), the numbers of primary ovoid (P)-type nerve fibers and demyelination (D)-type fibers in each image were counted.
8
Dehydration and Embedding Protocol for Electron Microscopy
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After concluding the staining procedure the sample was dehydrated by immersing it for 30 min each at 4°C in a graded ethanol (Serva, Germany) series of 50% ethanol in ultrapure water, 75% ethanol in ultrapure water and 100% ethanol, respectively. The sample was then immersed three times in acetone (Serva, Germany) 30 min each at room temperature. After completing the dehydration steps, the sample was infiltrated with a 1:1 mixture of acetone and Spurr’s resin (Sigma Aldrich, USA) with a component ratio of 4.1 g ERL 4221, 0.95 g DER 736 and 5.9 g NSA and 1% DMAE at room temperature for 12 hr on a 45 degree rotator in open 2 ml reaction tubes (Eppendorf, Germany). Infiltrated samples were then incubated in pure resin for 6 hr, placed in embedding molds and cured in a preheated oven at 70°C for 48–72 hr.
9
Transmission Electron Microscopy of Chlamydia trachomatis
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C. trachomatis infected HaCaT cells grown on 12 mm Nunc™ Thermanox™ coverslips (ThermoFisher, USA) were fixed with 2% glutaraldehyde in EMEM (BioWhittakerTM, Walkersville, USA), followed by two washing steps with EMEM and one step with 0.1M sodium cacodylate buffer, pH 7.4. The cells were fixed with 1% osmium tetraoxide, dehydrated with a graded series of ethanol (50, 70, 90, and 100%) at 24°C, infiltrated, and embedded in Spurr’s resin (Sigma, Steinheim, Germany) overnight at 60°C (S1 Table ). A beam capsule filled with Spurr resin was used to embed the fixed cells for TEM. Ultrathin sections (50–60 nm) were cut and collected onto 3.05 mm diameter, square uncoated mesh copper TEM grids, then double-stained with uranyl acetate and Reynold’s lead citrate (Sigma, Steinheim, Germany) for 10 minutes each [25 (link)]. Sections were viewed using a Jeol 1010 transmission electron microscope (JEOL Ltd) at an accelerating voltage of 100 kV. The TEM was interfaced with a Megaview III Software Imaging Systems camera unit (Soft Imaging System, Münster, Germany). Images were captured digitally, and measurements performed using iTEM analySIS (Soft Imaging System, Münster, Germany) image analyzing software.
10
Ultrastructural Analysis of Cells
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The cells on a carbon-coated aluminum sheet of 10 µm thickness with a stainless ring folder were agar-overlaid and quickly frozen in liquid nitrogen-solid nitrogen slush (frozen liquid nitrogen in a vacuum) at −210 °C. The frozen cells were subjected to freeze-substitution with 1% OsO4 in acetone at −80 °C for 2 days, dehydrated in absolute acetone at room temperature, substituted with propylene oxide, and then embedded in Spurr’s resin (Sigma-Aldrich). After the resin was cured by a specified method, the aluminum sheet was removed. The cells were serially sectioned at a thickness of 50–60 nm using an ultramicrotome (Leica EM UC7) and stained with 1% uranyl acetate followed by Reynold’s lead citrate54 (link). They were observed under electron microscope (Quanta 3D, FEI or CM-120, Philips).
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