Mouse heart samples were removed and fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). Heart samples were then rinsed with 0.1 M cacodylate buffer (pH 7.4) twice before post-fixation in 1% osmium tetroxide for 1 hour. After additional buffer rinses, heart samples were dehydrated through an ethanol series to 100% ethanol, infiltrated with a mixture of 100% propylene oxide and Eponate 12 resin (Ted Pella Inc., Redding, CA), followed by pure Eponate 12 resin overnight. Heart samples were embedded in beem capsules and placed in a 60°C oven for polymerization. Ultrathin sections were cut on a Leica UltraCut microtome at 70–80 nm and placed on 200 mesh copper grids. Sections were then stained with 5% uranyl acetate for 15 minutes followed by 2% lead citrate for 15 minutes. Samples were imaged with a JEOL JEM-1400 transmission electron microscope (Tokyo, Japan) equipped with a Gatan US1000 CCD camera (Pleasanton, CA). Mitochondrial mass and lipid content were measured using Image J software (National Institute of Health).
Eponate 12 resin
Manufactured by Ted Pella
Sourced in United States, Canada
Eponate 12 resin is a low viscosity epoxy resin primarily used for embedding and preparing samples for electron microscopy. It is a two-component system that cures at elevated temperatures to produce a hard, durable embedding medium. The resin is designed to provide high-quality sectioning and preservation of ultrastructural detail in specimens.
Automatically generated - may contain errors
Lab products found in correlation
Aqueous uranyl acetate, by Ted Pella (64 mentions)
Osmium tetroxide, by Polysciences (62 mentions)
1200 ex transmission electron microscope, by JEOL (61 mentions)
Leica ultracut uct ultramicrotome, by Leica Microsystems (54 mentions)
Amt 8 megapixel digital camera, by Advanced Microscopy Techniques (37 mentions)
Amt image capture engine v602, by Advanced Microscopy Techniques (35 mentions)
Uranyl acetate, by JEOL (28 mentions)
Paraformaldehyde 2.5 glutaraldehyde, by Polysciences (26 mentions)
Glutaraldehyde, by Polysciences (22 mentions)
Paraformaldehyde (pfa), by Polysciences (19 mentions)
Osmium tetroxide, by Ted Pella (9 mentions)
Paraformaldehyde 2.5 glutaraldehyde, by Ted Pella (8 mentions)
8 megapixel digital camera, by Advanced Microscopy Techniques (7 mentions)
Leica ultracut uc7 ultramicrotome, by Leica Microsystems (6 mentions)
Jem 1400 tem, by JEOL (6 mentions)
Ultracut s ultramicrotome, by Leica camera (5 mentions)
Uranyl acetate, by Leica Microsystems (5 mentions)
Us1000 ccd camera, by Ametek (4 mentions)
Leica ultracut s ultramicrotome, by Leica Microsystems (4 mentions)
Gatan us1000 2k 2k ccd camera, by Ametek (4 mentions)
148 protocols using eponate 12 resin
1
Ultrastructural Analysis of Mouse Hearts
2
Ultrastructural Analysis of Monolayer Cells
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Monolayer cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). Cells were then rinsed with 0.1 M cacodylate buffer (pH 7.4) twice before post-fixation in 1% osmium tetroxide for 1 hour. After additional buffer rinses, cells were dehydrated through an ethanol series to 100% ethanol. Cells were infiltrated with a mixture of 100% ethanol and Eponate 12 resin (Ted Pella Inc., Redding, CA), and then pure Eponate 12 resin overnight. Cells were embedded in multiwall plate and then placed in a 60 °C oven for polymerization. Ultrathin sections were cut on a Leica UltraCut microtome at 70–80 nm and placed on 200 mesh copper grids. Sections were then stained with 5% uranyl acetate for 15 minutes followed by 2% lead citrate for 15 minutes. Cells were imaged with a JEOL JEM-1400 transmission electron microscope (Tokyo, Japan) equipped with a Gatan US1000 CCD camera (Pleasanton, CA) by a blinded observer. At least 10 high-powered images (15,000x, 18 μm2) were examined and various variables were quantified with software analysis using Image J software (National Institutes of Health, Bethesda MD).
3
Mitochondrial Imaging in T-cells
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Mito Tracker Green (Life Technologies) was used to measure mitochondrial mass. In vitro Rapa-treated (Rapa+) and untreated (Rapa−) T-cells were stained with 50 nM MitoTracker Green or 10 μM TMRM for 15 min at 37 °C, according to the manufacturer’s manual. Stained cells were washed three times with PBS, followed by incubation with 5 μg/mL of Hoechst 33342 solution (Life Technologies) for 20 min at room temperature. Confocal images were acquired using the Zeiss LSM700 confocal microscope (Carl Zeiss, Oberkochen, BW, Germany) with ×20 objective [45 (link)]. For electron microscope imaging, cell pellets (2 × 106 T-cells/each) were fixed in 2% paraformaldehyde and 2.5% glutaraldehyde in 100 mM sodium cocodylate. After fixation, samples were washed in cacodylate buffer and fixed in 1% osmium tetroxide. After extensive washing in H2O, samples were stained with 1% aqueous uranyl acetate for 1 h and washed again. Samples were dehydrated in ethanol, embedded in Eponate 12™ resin (Ted Pella, Redding, CA, USA), and sectioned for imaging [70 (link)]. Images were acquired using a JOEL 1200 EX transmission electron microscope.
4
Ultrastructural Analysis of Plasmodium-Infected RBCs
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For ultrastructural analysis, Plasmodium-infected red blood cells were fixed in 1% glutaraldehyde (Polysciences Inc., Warrington, PA)/1% osmium tetroxide (Polysciences Inc.) in 50 mM phosphate buffer, pH 7.2 for 1 hr at 4°C. This low osmolarity fixation was used to remove dense, soluble cytoplasmic components, allowing unobscured membrane analysis. Cells were washed in phosphate buffer, rinsed extensively in dH2O prior to en bloc staining with 1% aqueous uranyl acetate (Ted Pella Inc., Redding, CA) for 1 hr. Following several rinses in dH2O, samples were dehydrated in a graded series of ethanol and embedded in Eponate 12 resin (Ted Pella, Inc.). Sections of 90 nm were cut, stained with uranyl acetate and lead citrate, and viewed on a JEOL 1200 EX transmission electron microscope (JEOL USA, Peabody, MA) at an accelerating voltage of 80kV.
5
Ultrastructural Analysis of Cells
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For ultrastructural analysis, cells were fixed for 1 h at 22 °C in 2% paraformaldehyde, 2.5% glutaraldehyde (Polysciences), and 0.05% malachite green (Sigma-Aldrich) in 100 mM sodium cacodylate buffer (pH 7.2). malachite green was incorporated into the fixative for stabilization of lipid constituents soluble in aqueous glutaraldehyde. Samples were washed in cacodylate buffer and were post-fixed for 1 h in 1% osmium tetroxide (Polysciences). Samples were then rinsed extensively in distilled water before en bloc staining for 1 h with 1% aqueous uranyl acetate (Ted Pella). Following several rinses in distilled water, the samples were dehydrated in a graded series of ethanol and embedded in Eponate 12 resin (Ted Pella). Sections 95 nm in thickness were cut with a Leica Ultracut UC7 ultramicrotome (Leica Microsystems), then stained with uranyl acetate and lead citrate and viewed on a Tecnai G2 Spirit BioTWIN transmission electron microscope (FEI) at 60 kV.
6
Mitochondrial Ultrastructural Analysis
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Samples were fixed in 2% paraformaldehyde/2.5% glutaraldehyde (Polysciences) in 100 mM sodium cacodylate buffer, pH 7.2 for 1 hr at room temperature. Samples were washed in sodium cacodylate buffer and postfixed in 1% osmium tetroxide (Polysciences) for 1 hr. Next, samples were rinsed in dH2O prior to en bloc staining with 1% aqueous uranyl acetate (Ted Pella) for 1 hr. Following several rinses in dH2O, samples were dehydrated in a graded series of ethanol and embedded in Eponate 12 resin (Ted Pella). Sections of 95 nm were cut with a Leica Ultracut UCT ultramicrotome (Leica Microsystems), stained with uranyl acetate and lead citrate, and viewed on a JEOL 1200 EX transmission electron microscope (JEOL USA) equipped with an AMT eight megapixel digital camera and AMT Image Capture Engine V602 software (Advanced Microscopy Techniques). The length of 100 random mitochondria for each condition were measured and plotted.
7
Ultrastructural Analysis of Cells
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Cells incubated with µP-3-Tf-A488 were fixed for 25 min in 2% PFA and 2.5% glutaraldehyde (EM grade, Merck, Darmstadt, Germany) in 0.1 M cacodylate buffer (Sigma-Aldrich). They were post-fixed with 1% osmium tetroxide (TAAB Laboratories Equipment Ltd, Aldermaston, England) for 2 h and dehydrated in a graded ethanol series (15 min in 30%, 30 min in 50%, 30 min in 90%, 30 min in 95% and twice 30 min in 100%), before embedding the samples in Eponate 12 resin (TED Pella Inc., Redding, USA). Polymerization was performed at 60 °C for 48 h. Ultrathin sections of 70 nm were obtained using a Leica ultracut microtome (Leica Microsystems, Wetzlar, Germany) and placed on 200 mesh copper grids. Finally, samples were contrasted with a 2% uranyl acetate solution (Sigma-Aldrich) for 30 min and subsequently with a Reynolds lead citrate solution for 5 min and observed under a TEM Jeol JEM-1400 (Jeol Ltd, Tokyo, Japan).
8
Localization of FimH on Intestinal Epithelium
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For localization of FimH on intestinal epithelium, ETEC infected CIE were fixed in 2% paraformaldehyde/0.02% glutaraldehyde (Polysciences Inc., Warrington, PA) in 100 mM PIPES/0.5 mM MgCl2, pH 7.2 for 1 h at 4°C. Samples were washed with PIPES buffer, blocked with 5% Fetal Bovine Serum /5% Normal Goat Serum for 20 min and subsequently incubated with rabbit anti-FimH antibody for 1 h, followed by secondary goat anti-rabbit antibody conjugated to 18 nm colloidal gold for 1 h. Samples were washed in buffer and postfixed in 1% osmium tetroxide (Polysciences Inc.) for 1 h. Samples were then rinsed extensively in dH2O prior to en bloc staining with 1% aqueous uranyl acetate (Ted Pella Inc., Redding, CA) for 1 h. Following several rinses in dH2O, samples were dehydrated in a graded series of ethanol and embedded in Eponate 12 resin (Ted Pella Inc.). Sections of 95 nm were cut with a Leica Ultracut UCT ultramicrotome (Leica Microsystems Inc., Bannockburn, IL), stained with uranyl acetate and lead citrate, and viewed on a JEOL 1200 EX transmission electron microscope (JEOL USA Inc., Peabody, MA) equipped with an AMT 8 megapixel digital camera (Advanced Microscopy Techniques, Woburn, MA).
9
Ultrastructural Analysis of Biological Samples
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For ultrastructural analyses, samples were fixed in 2% paraformaldehyde/2.5% glutaraldehyde (Polysciences, Warrington, PA) in 100 mM sodium cacodylate buffer, pH 7.2 for 2 hours at room temperature and then overnight at 4°C. Samples were washed in sodium cacodylate buffer at room temperature and postfixed in 1% osmium tetroxide (Polysciences) for 1 hour. Samples were then rinsed extensively in dH20 prior to en bloc staining with 1% aqueous uranyl acetate (Ted Pella, Redding, CA) for 1 hour. Following several rinses in dH20, samples were dehydrated in a graded series of ethanol and embedded in Eponate 12 resin (Ted Pella). Sections of 95 nm were cut with a Leica Ultracut UCT ultramicrotome (Leica Microsystems, Bannockburn, IL), stained with uranyl acetate and lead citrate, and viewed on a JEOL 1200 EX transmission electron microscope (JEOL USA, Peabody, MA) equipped with an AMT 8-MP digital camera and AMT Image Capture Engine V602 software (Advanced Microscopy Techniques, Woburn, MA).
10
Electron Microscopy Ultrastructural Analysis
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Electron microscopy images were obtained from the Molecular Microbiology Imaging Facility. For ultrastructural analysis, samples were fixed in 2% paraformaldehyde/2.5% glutaraldehyde (Polysciences Inc.) in 100 mmol/L cacodylate buffer (pH 7.2) for 1 h at room temperature. Samples were washed in cacodylate buffer and postfixed (1 h) in 1% osmium tetroxide (Polysciences Inc.). Samples were rinsed extensively in deionized H2O before en bloc staining (1 h) with 1% aqueous uranyl acetate (Ted Pella Inc.). After several rinses, the samples were dehydrated in a graded series of ethanol and embedded in Eponate 12 resin (Ted Pella Inc.). Sections (95 nm) (Ultracut UCT ultramicrotome; Leica Microsystems Inc.) were stained with uranyl acetate and lead citrate and viewed on a JEOL 1200 EX transmission electron microscope (JEOL USA Inc.), equipped with an 8-megapixel digital camera (Advanced Microscopy Techniques).
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