To characterise the morphology of MOX-loaded SLNs and NLCs, the particles were observed under a FEI Tecnai G2 Spirit BioTwin (Thermo Fisher, Waltham, MA, USA) transmission electron microscope (TEM) operating at 40 kV and 80,000× magnification. Samples were mounted on copper grids with a mesh size of 200 (75 microns), stained with 2 % uranyl acetate for 2 min, and then the excess removed with filter paper. The remainder was dried in a Petri dish for 2 h before microscopy.
Fei tecnai g2 spirit biotwin
Manufactured by Thermo Fisher Scientific
Sourced in United States
The FEI Tecnai G2 Spirit BioTwin is a transmission electron microscope designed for high-resolution imaging of biological samples. It is capable of providing detailed structural information about cellular components, proteins, and other nanoscale biological structures.
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Lab products found in correlation
Zetaview, by Particle Metrix (2 mentions)
Ultracut uc6, by Leica (1 mentions)
Durcupan resin, by Merck Group (1 mentions)
Morada, by Olympus (1 mentions)
Uc7 ultramicrotome, by Leica (1 mentions)
S 4800, by Hitachi (1 mentions)
E 1045, by Hitachi (1 mentions)
Sw32ti, by Beckman Coulter (1 mentions)
Epoxy resin, by Merck Group (1 mentions)
Flow nanoanalyzer, by NanoFCM (1 mentions)
Gatan digital imaging system, by Ametek (1 mentions)
Gatan microscopy suite, by Ametek (1 mentions)
Whatman 1 filter paper, by Cytiva (1 mentions)
Em uc7 ultramicrotome, by Leica (1 mentions)
15 protocols using fei tecnai g2 spirit biotwin
1
Characterization of MOX-loaded SLNs and NLCs
2
Ultrastructural Analysis of Gastrocnemius Muscle
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Fresh gastrocnemius muscles were fixed overnight at room temperature in 2.5 % glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer and then 72 h at 4ºC before processing for electron microscopy. Muscle samples were post-fixed with 2% osmium, rinsed, dehydrated and embedded in Durcupan resin (Fluka, Sigma-Aldrich, St. Louis, USA). To verify orientation, serial semithin sections (1.5 mm) were cut with an Ultracut UC-6 (Leica microsystems, Wetzlar, Germany) and mounted into slides and stained with 1% toluidine blue. In selected correct orientation samples, ultrathin sections (0.07 – 0.08 mm) were cut using an Ultra 45º diamond knife (Diatome Ltd, Nidau, Switzerland) and stained with lead citrate. Finally, photomicrographs were obtained under a transmission electron microscope FEI Tecnai G2 Spirit BioTwin (ThermoFisher Scientific company, Oregon, USA), using a digital camera Morada (Olympus Soft Image Solutions GmbH, Münster, Germany).
3
Immunogold-Electron Microscopy for MIB2/DLL3 or MIB2/Gm364 Co-Labeling
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For MIB2/DLL3 or MIB2/Gm364 co-labeling and signal acquisition through Immunogold-electron microscopy, 50–100 oocytes per repeat were blocked with 1% BSA, incubated with MIB2 Ab at 4 °C overnight, next labeled with Donkey anti-rabbit IgG/15 nm-Gold at room temperature for 2 h. Then oocytes were re-blocked with 1% BSA (to eliminate the cross-reaction between two antibodies), incubated with DLL3 or Gm364 Ab (4 °C, overnight), and then labeled with Donkey anti-rabbit IgG/35 nm-Gold or 10 nm-Gold at room temperature for 2 h.
After being labeled, oocytes were fixed in 2.5% glutaraldehyde for 2 h at 4 °C, washed three times with PBS, and stained with eosin for 2 min to facilitate the oocyte positioning by eye. Oocytes were then placed in 2% agarose and spun for 5 min at 13,000 rpm and held overnight at 4 °C. The next day, the agarose piece with eosin-stained oocytes (red in the piece) was trimmed and sent to the Testing and Analysis Center (Nanjing Medical University) for sample preparation for transmission electron microscopy. Electron microscopy pictures were obtained with a transmission electron microscope (FEI Tecnai G2 Spirit Bio Twin; Thermo Fisher Scientific).
After being labeled, oocytes were fixed in 2.5% glutaraldehyde for 2 h at 4 °C, washed three times with PBS, and stained with eosin for 2 min to facilitate the oocyte positioning by eye. Oocytes were then placed in 2% agarose and spun for 5 min at 13,000 rpm and held overnight at 4 °C. The next day, the agarose piece with eosin-stained oocytes (red in the piece) was trimmed and sent to the Testing and Analysis Center (Nanjing Medical University) for sample preparation for transmission electron microscopy. Electron microscopy pictures were obtained with a transmission electron microscope (FEI Tecnai G2 Spirit Bio Twin; Thermo Fisher Scientific).
4
Ultrastructural Analysis of Autophagic Lysosomes
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The cells were fixed in 2.5% glutaraldehyde solution at 4 °C for 4 h, rinsed four times using 0.1 M phosphate buffer saline (PBS) (15 min each), and fixed with 1% osmic acid solution at 4 °C for 2 h. The cells were subsequently dehydrated using 50, 70, 90, and 100% gradient ethanol (15 min each) and permeabilized (100% acetone: resin = 1:1 for 2 h, 100% acetone: resin = 1:2 for 2 h, pure resin overnight). The cells were then embedded in Epon812 resin, aggregated in an oven (37°C for 12 h, 45°C for 12 h, and 60°C for 48 h), sectioned into ultrathin slices (thickness, 60–70 nm), stained with uranyl acetate and lead nitrate and observed under a TEM (FEI Tecnai G2 Spirit Bio TWIN, Thermo Fisher Scientific, Waltham, MA, USA). Five visual fields were randomly selected to observe and quantify the number of autophagic lysosomes. The experiment was repeated three times.
5
Ultrastructural Localization of Fam70A and Wnt5a
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Metaphase I oocytes were blocked with 1% BSA, incubated with Fam70A Ab (4°C, overnight) and next labelled with Donkey anti‐rabbit IgG/Gold (35 nm, room temperature for 2 hours). Then, oocytes were re‐blocked with 1% BSA (to eliminate the cross‐reaction between two antibodies), incubated with Wnt5a Ab (4°C, overnight) and then labelled with Donkey anti‐rabbit IgG/Gold (15 nm, room temperature for 2 hours).
After being labelled, oocytes were fixed in 2.5% glutaraldehyde for 2 hours at 4°C, washed three times with PBS and stained with eosin for 2 minutes to facilitate the oocyte positioning by eye. Oocytes were then placed in 2% agarose and spun for 5 minutes at 13 000 rpm and held overnight at 4°C. The next day, the agarose piece with eosin‐stained oocytes (red in the piece) was trimmed and sent to the Testing and Analysis Center (Nanjing Medical University) for sample preparation for transmission electron microscopy. Electron microscopy pictures were obtained with a transmission electron microscope (FEI Tecnai G2 Spirit Bio Twin; Thermo Fisher Scientific).
After being labelled, oocytes were fixed in 2.5% glutaraldehyde for 2 hours at 4°C, washed three times with PBS and stained with eosin for 2 minutes to facilitate the oocyte positioning by eye. Oocytes were then placed in 2% agarose and spun for 5 minutes at 13 000 rpm and held overnight at 4°C. The next day, the agarose piece with eosin‐stained oocytes (red in the piece) was trimmed and sent to the Testing and Analysis Center (Nanjing Medical University) for sample preparation for transmission electron microscopy. Electron microscopy pictures were obtained with a transmission electron microscope (FEI Tecnai G2 Spirit Bio Twin; Thermo Fisher Scientific).
6
Ultrastructural Analysis of Leaf Cells
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Fresh and fully expanded leaves from wt-WD1 and yl2.1 seedlings were cut into small pieces and fixed with 1 mL of 0.1 M glutaraldehyde in a phosphate buffer at 4 °C for 12 h before they were placed in a vacuum at room temperature for 30 min, then rinsed and fixed with 1.0% osmium tetroxide at 4 °C for 1.5 h. The samples were dehydrated by a graded ethanol series and finally embedded in Epon 812 resin (Shell Chemical, America). Thin sections were sectioned with a Leica UC-7 ultra-microtome (Leica, Germany) and stained in uranyl acetate and lead citrate. Finally, the stained samples were viewed under a FEI Tecnai G2 Spirit Biotwin (Thermo Fisher Scientific, Shanghai, China) transmission electron microscope.
7
Electron Microscopy of Extracellular Vesicles
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To analyze EV morphology, EVs were resuspended in PBS, and 6 μL were placed on a carbon-coated grid and contrasted with 2% uranyl acetate. EVs were then observed under an FEI Tecnai G2 Spirit BioTwin (Thermo Fisher Scientific, Oregon, USA) transmission electron microscope. Images obtained from each sample were examined by 3 observers, and artifacts were quantified based on their colour, size and shape.
8
Visualizing APEC Outer Membrane Vesicles
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APEC_OMVs were visualized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). For SEM visualization, 10 μL of the purified APEC_OMVs was dropped on a 5 mm × 5 mm silicon slice, dried at 20 °C and sputter-coated with gold-palladium using an ion-sputtering coater (E-1045; Hitachi, Tokyo, Japan). The prepared nanovesicles were observed using a Field Emission Scanning Electron Microscope (S-4800, Hitachi, Tokyo, Japan). For TEM visualization, 200 μg/mL of the nanovesicles was adhered to 300-mesh copper grids for 10 min followed by negatively staining with 1% phosphotungstic acid (pH 7.2), and then viewed using FEI Tecnai™ G2 Spirit BioTWIN (FEI Company, Hillsboro, OR, USA) at 100 kV.
9
Liposome Morphology Characterization
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Liposomes were diluted (1:50) with deionized water, and 1 drop of the formulation was applied onto a carbon-coated copper grid. After drying and adhesion, samples were negatively stained by 2% phosphotungstic acid and analyzed with a 120-kV transmission electron microscope (FEI Tecnai G2 Spirit BioTwin; FEI Company, Hillsboro, OR, USA).
10
Isolation and Characterization of BMSC-Derived Exosomes
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When passage 3 BMSCs reached a confluency of 80–90%, the culture medium without exosomes was replaced, and the supernatant was collected after 2 days. The exosomes in the supernatant of BMSCs were extracted by differential ultracentrifugation (Rotor: SW 32 Ti, Beckman Coulter, Brea, CA, USA). Briefly, the supernatant was collected by centrifugation at 300, 2000, and 10,000 × g for 10, 10, and 30 min to remove the cells and cell debris. Next, protein contaminants were removed by ultracentrifugation of the supernatant at 110,000 × g for 70 min twice, and the precipitate was resuspended in PBS buffer. Subsequently, exosomes in the precipitate were resuspended in 100 µL PBS and stored at −80 ° C (Théry et al., 2006 ; Chen et al., 2019 (link)).
The approximate concentration and diameter of the exosomes were measured by nanoparticle tracking analysis (NTA) using a ZetaView Particle Metrix (Particle Metrix, Meerbusch, Germany). The shape and size of the exosomes were observed by transmission electron microscopy (TEM) (FEI Tecnai G2 Spirit BioTwin; FEI, Hillsboro, OR, USA). The exosome positive markers (CD81, Hsp70, and TSG101) and purity control (Calnexin) were detected by Western blotting.
The approximate concentration and diameter of the exosomes were measured by nanoparticle tracking analysis (NTA) using a ZetaView Particle Metrix (Particle Metrix, Meerbusch, Germany). The shape and size of the exosomes were observed by transmission electron microscopy (TEM) (FEI Tecnai G2 Spirit BioTwin; FEI, Hillsboro, OR, USA). The exosome positive markers (CD81, Hsp70, and TSG101) and purity control (Calnexin) were detected by Western blotting.
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