Heart and hind-limb skeletal muscle were excised and fixed in phosphate buffered 4% formaldehyde with 1% glutaraldehyde. The samples were rinsed in 0.1M phosphate buffer and then post-fixed with 1% Zetterqvist’s buffered Osmium Tetroxide for 30 minutes. The samples were dehydrated in 70% alcohol for 10 minutes and then in 95% alcohol for 10 minutes. The samples were then twice dehydrated in 100% alcohol for 10 minutes and then twice dehydrated in propylene oxide for 10 minutes, for each dehydration. The samples were resin infiltrated first in a 1:1 propylene oxide/resin mix for 30 minutes and then in 100% resin for 30 minutes under 25psi vacuum. The samples were then flat-embedded in a BEEM capsule and filled to the top and polymerized at 85°C for 90 minutes. The embedded samples were then sectioned and stained with a drop of uranyl acetate and microwaved for 30 seconds. The section was then gently dipped into distilled water and placed on filter paper. A drop of Reynold’s lead citrate stain was then placed on the section and microwaved for 20 seconds and then rinsed in distilled water. The section was then photographed at 20,000× using a JEOL 1230 transmission electron microscope with AMT imaging software. Five randomly chosen images per animal (n=3) were quantified for mitochondrial number and area using NIS-Elements BR imaging software.
1230 transmission electron microscope
Manufactured by JEOL
Sourced in Japan, United States
The JEOL 1230 is a transmission electron microscope designed for high-resolution imaging and analysis of materials at the nanoscale level. It is equipped with a LaB6 electron source and provides a maximum accelerating voltage of 120 kV. The instrument can achieve a resolution of 0.2 nm and is capable of performing various imaging modes, including bright-field, dark-field, and high-resolution imaging. The JEOL 1230 is a versatile tool for researchers and scientists working in material science, nanotechnology, and life sciences.
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Lab products found in correlation
Epon 12, by Ted Pella (3 mentions)
Amt 2k digital camera, by JEOL (2 mentions)
Dulbecco modified eagle medium (dmem), by Gemini Bio (2 mentions)
Delipidized fetal bovine serum, by Gemini Bio (2 mentions)
Carbon coated em grids, by Electron Microscopy Sciences (2 mentions)
Epon 812, by Electron Microscopy Sciences (1 mentions)
Ls230, by Beckman Coulter (1 mentions)
Miniflex diffractometer, by Rigaku (1 mentions)
Nano z, by Malvern Panalytical (1 mentions)
Anti l1cam antibody, by Abcam (1 mentions)
Uranyl acetate, by Electron Microscopy Sciences (1 mentions)
Mpms 5xl squid magnetometer, by Quantum Design (1 mentions)
Tga sdta 851eapparatus, by Mettler Toledo (1 mentions)
Leica uct ultramicrotome, by Leica Microsystems (1 mentions)
7150 automatic biochemical analyzer, by Hitachi (1 mentions)
Accu chek blood glucose meter, by Roche (1 mentions)
Sds page electrophoresis, by Bio-Rad (1 mentions)
Goat anti rabbit antibody, by Boster Bio (1 mentions)
Streptozotocin (stz), by Merck Group (1 mentions)
Ultracut e ultramicrotome, by Leica camera (1 mentions)
58 protocols using 1230 transmission electron microscope
1
Transmission Electron Microscopy of Skeletal Muscle
2
Ultrastructural Analysis of Mouse Mesenteric Arteries
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Third-order mesenteric arteries were dissected from mice and fixed in 4% paraformaldehyde/2.5% glutaraldehyde/PBS for a minimum of 4 hours. Arteries were then processed at the UVA Advanced Microscopy Facility. The arteries were washed in cacodylate buffer, incubated in 2% osmium tetroxide for 1 hour, washed in cacodylate buffer, and dehydrated with ethanol washes prior to incubation in 1:1 propylene oxide/epoxy resin (PO/EPON) overnight. The next day, the samples were incubated in a 1:2 PO/EPON mixture for 2 hours, a 1:4 PO/EPON mixture for 4 hours, and 100% EPON overnight. The next day, the samples were baked in an oven at 65°C prior to sectioning. Ultrathin 70 nm sections were mounted on a mesh copper grid. Sections were contrast stained with 0.25% lead citrate for 5 minutes, 2% uranyl acetate for 20 minutes, and then again 0.25% lead citrate for 5 minutes. Sections were visualized and imaged using a JEOL 1230 Transmission Electron Microscope. To avoid the possibility of double counting an HIEL, only 1 section per 5 μm artery length was analyzed, and a minimum of 500 μm of IEL length was analyzed per artery.
3
Prion Protein Fibrilization Imaging
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For electron microscopy, samples were applied to carbon coated copper mesh grids (Ted Pella, inc.). In detail 5 μl aliquots PrP samples subjected to fibrillation conditions in the absence or presence of seeds for 24 h (HuPrP, FePrP, CaPrP, PoPrP, MoPrP, HaPrP) or 48 h (BoPrP) was applied for 2 min. The grid was blotted dry and rinsed with 5 μl of dH2O, followed by counterstaining with 5 μl of with 2% (w/v) uranyl acetate dissolved in dH2O for 30 s. Micrographs were collected using a Jeol 1230 transmission electron microscope operating at 100 kV equipped with a CCD camera.
4
Immunogold Labeling of Viral Particles
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Pelleted viral fractions from the KT-glycerol gradients were resuspended in 200 μl of phosphate-buffered saline (PBS) after a short (5-s) sonication burst. Next, 25 μl of this material was dropped onto carbon-coated transmission electron microscopy (TEM) grids (Formvar-coated nickel TEM grids that were carbon coated) and incubated at room temperature (RT) for 1 h, with occasional mixing by movement of the grids in the drop. Washing of TEM grids was done by briefly dipping the grids into water and then suspending the grids on drops of water (25 μl). After adsorption of viral material onto the grids, the grids were washed and then blocked in 5% nonfat milk with 5% normal goat serum, and the grids were incubated in drops of primary antibody (1:50 dilution) at RT for 4 h and then overnight at 4°C. After washing, the grids were incubated with secondary immunogold antibody (goat anti-mouse or goat anti-rabbit ultrasmall gold beads; 1:25 dilution) for 4 h at RT. After washing, the grids were suspended in freshly prepared silver enhancement solution (Aurion) for 30 min, washed with water, and then negatively stained with 2.5% uranium acetate drops for 2 min at RT. The uranyl acetate was wicked off with filter paper, and the grids were allowed to air dry overnight. Grids were viewed with a JEOL 1230 transmission electron microscope.
5
Ultrastructural Analysis of Endocytic Pathways
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Briefly, 14 × 107 wild-type trophozoites, glvps4a-ha, glvps4a:as, glvps4aE228Q-ha, glrab11–ha, or ds-glrab11 transgenic trophozoites were grown in axenic culture at 37 °C in 8-ml tubes containing TYI-S-33 medium until monolayer and washed twice with warm PBS 1X (37 °C) and adherent cells fixed with a solution containing 2.5% glutaraldehyde, 4% paraformaldehyde, and sucrose 4% in 0.1 M cacodylate buffer. Then, the cells were scraped off the tube wall with a rubber policeman; washed in 0.1 M cacodylate buffer; and postfixed for 60 min at room temperature in a solution containing 1% osmium tetroxide, 0.8% potassium ferrocyanide, and 5 mM CaCl2 in 0.1 M cacodylate buffer. Afterward, the cells were washed in buffer, dehydrated in acetone, and embedded in Polybed resin. Thin sections were stained with uranyl acetate and lead citrate and observed in a JEOL 1230 Transmission Electron Microscope.
6
Neutrophil-Derived Vesicles Interact with Tumor Cells
7
Ultrastructural Analysis of Utricle Junctions
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Transmission electron microscopy (TEM) images of the apical junctional region of utricles from Actn4 KO mice were obtained as previously described (Burns et al., 2008 (link)). Briefly, mice were euthanized as above, and within 3 min a fixative solution composed of 3% glutaraldehyde in 0.15 M sodium cacodylate buffer at pH 7.4 was injected into the superior semicircular canal to replace the endolymph. The labyrinth was then isolated and fixed overnight at 4°C. Then the utricle was dissected in 0.15 M cacodylate buffer and post-fixed in 1% osmium-tetroxide. The tissue was dehydrated in a graded ethanol series and infiltrated with Epon 812 (Electron Microscopy Sciences, Hatfield, PA, United States). Thin sections were cut in the plane parallel to the epithelial surface with a diamond knife. Sections were collected on copper grids and stained 5 min with lead citrate, 15 min with uranyl acetate, and 5 min with lead citrate. A JEOL 1230 transmission electron microscope was used for imaging.
8
Analysis of Trophozoite Interactions with bLF and bLFcin
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4 × 107wild-type trophozoites were incubated with 12.5 µM of bLF or 2.6 µM of bLFcin for 30 min at 37 °C. The trophozoites were washed twice with PBS at 37 °C and then fixed in a solution containing 2.5% glutaraldehyde, 4% paraformaldehyde, and sucrose 4% in 0.1 M cacodylate buffer. The cells were centrifuged for 5 min at 600 × g, washed in 0.1 M cacodylate buffer and postfixed for 60 min at room temperature in a solution containing 1% osmium tetroxide, 0.8% potassium ferrocyanide, and 5 mM CaCl2 in 0.1 M cacodylate buffer. Subsequently, the cells were washed in buffer, dehydrated in acetone and embedded in Polybed resin. Thin sections were stained with uranyl acetate and lead citrate and observed in a JEOL 1230 Transmission Electron Microscope.
9
Ultrastructural Analysis of Mouse Tissues
10
Electron Microscopy Sample Preparation
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The samples (membrane particles bound to SAX beads) were fixed in 2% glutaraldehyde/0.1 M sodium cacodylate, pH 7.2 overnight at 4°C, and then postfixed in 2% OsO4 buffered with 100 mM sodium cacodylate, pH 7.2 overnight at room temperature. The samples were then washed with water, dehydrated in an increasing ethanol series (30%, 50%, 60%, 70%, 80%, 90%, 98% for 10 minutes each followed by 100% × 3 for 10 minutes) and then infiltrated stepwise from ethanol into Spurr’s resin (2:1, 1:1, 1:2 for 30 min each followed by 100% Spurr’s x 3 overnight) and then embedded and baked at 60°C for 2 days. Ultra-thin sections were cut from each sample, collected on grids, and imaged on a JEOL 1230 Transmission Electron Microscope (Peabody, MA).
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