Exosomes pellet was fixed in 2% glutaraldehyde in Sorensen buffer (pH 7.4) for 2 h, post-fixed in 1% osmium tetroxide (OsO4) in aqueous solution for 2 h, dehydrated in graded concentrations of acetone and embedded in Epon–Araldite mixture (Electron Microscopy Sciences, Fort Washington, PA, USA). The semithin sections (1 µm in thickness) were examined by light microscopy (Olympus BX51, Olympus Optical, Hamburg, Germany) and stained with toluidine blue. The ultrathin sections were cut at a 70 nm thickness, placed on Cu/Rh grids with Ultracut E (Reichert, Wien, Austria), and observed with TEM using a Morgagni 268D electron microscope (Philips).
Morgagni 268d electron microscope
Manufactured by Philips
Sourced in Austria, United States, Netherlands
The Morgagni 268D is an electron microscope designed for high-resolution imaging of samples. It features a tungsten filament and a LaB6 electron source, providing a stable and reliable electron beam. The microscope operates at accelerating voltages up to 100 kV, enabling detailed examination of various materials and specimens.
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Lab products found in correlation
Epon araldite mixture, by Electron Microscopy Sciences (1 mentions)
Megaview 2 camera, by Olympus (1 mentions)
G box f3 genesys, by Syngene (1 mentions)
Hsp70, by Santa Cruz Biotechnology (1 mentions)
Ultracut e, by Reichert Technologies (1 mentions)
Bicinchoninic acid bca kit, by Thermo Fisher Scientific (1 mentions)
Zetasizer nano z, by Malvern Panalytical (1 mentions)
Optima le 80k, by Beckman Coulter (1 mentions)
Baf400d, by Oerlikon Balzers (1 mentions)
9 protocols using morgagni 268d electron microscope
1
Exosome Ultrastructural Characterization
2
Ultrastructural and Molecular Analysis of ASCs-Derived Exosomes
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Exosomes were fixed in 2% glutaraldehyde in DNase/RNase-Free Distilled Water (for 10 min on 150 mesh formvar and carbon-coated copper grids (Società Italiana Chinici, Rome, Italy), and dried under a hood. The images were acquired with a transmission electron microscopy (TEM) using a Morgagni 268D electron microscope (Philips, Andover, MA, United States) operating at 80 kV and equipped with a Megaview II camera (Olympus corporation, Tokyo, Japan) for digital image acquisition.
To perform Western blot of ASCs-exosomes, the proteins were denatured, separated on 4-12% polyacrylamide gels, transferred onto a nitrocellulose membrane and probed with antibodies against Alix (1:50, Santa Cruz Biotechnology, Q-19:sc-49268), heat shock protein 70 (HSP70, 1:100 Santa Cruz Biotechnology, sc-1060), and tetraspanine CD81 (1:100 Santa Cruz Biotechnology, sc-9158). The appropriate HRP-conjugated secondary antibodies against primary antibody (all secondary antibodies from Dako Agilent) were used. ASCs lysates were used as positive control. The blots were then incubated with a chemiluminescent HRP substrate and detected with G:BOX F3 GeneSys (Syngene, United Kingdom).
To perform Western blot of ASCs-exosomes, the proteins were denatured, separated on 4-12% polyacrylamide gels, transferred onto a nitrocellulose membrane and probed with antibodies against Alix (1:50, Santa Cruz Biotechnology, Q-19:sc-49268), heat shock protein 70 (HSP70, 1:100 Santa Cruz Biotechnology, sc-1060), and tetraspanine CD81 (1:100 Santa Cruz Biotechnology, sc-9158). The appropriate HRP-conjugated secondary antibodies against primary antibody (all secondary antibodies from Dako Agilent) were used. ASCs lysates were used as positive control. The blots were then incubated with a chemiluminescent HRP substrate and detected with G:BOX F3 GeneSys (Syngene, United Kingdom).
3
Ultrastructural Analysis of SW-480/Trophozoite Interactions
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After 15, 30, and 45 min interactions, SW-480/trophozoites co-cultures were washed twice with PBS to remove any unattached amoeba and fixed for 60 min with 2.5 % (v/v) glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, and postfixed for 60 min with 1 % (w/v) osmium tetroxide in the same buffer. After dehydration, with increasing concentrations of ethanol and propylene oxide, samples were embedded in Polybed epoxy resins and polymerized at 60°C for 24 h. Thin sections (60 nm) were obtained and stained with uranyl acetate and lead citrate for its examination in a Philips Morgagni 268 D electron microscope.
4
Electron Microscopy Sample Preparation
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Cells were grown in LB medium to an OD578 of 1, pelleted in a microcentrifuge tube and fixed in 4% glutaraldehyde in PBS for 2h at 4°C. After washing with PBS containing 0,25M Glucose, cells were treated with 1% OsO4 for 2,5 h at 4°C and washed with distilled H2O. Samples were dehydrated with an ethanol step gradient (50%, 70%, 90%, 100%, 10 min each) and a final acetone step (100%, 10 min). Dehydrated samples were treated for 2h with 50% “epon” [48 ] in acetone and pure epon for 4h. Polymerization was carried out at 60°C for 24h. 60–70 nm thin cuts were made on a Ultracut E (Reichert-Jung) and stained with 6% UrAc (1 h) and PbAc (2 min) according to Millonig [49 (link)]. Pictures were recorded on a Philips Morgagni 268D electron microscope at 80 kV.
5
Characterization of Therapeutic Exosomes
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cAd-MSCs and TK-cAd-MSCs exosomes were isolated by ultracentrifugation using 70 Ti rotor in an Optima LE-80K ultracentrifuge (Beckman Coulter) and characterized by transmission electron microscopy (TEM; Morgagni 268D electron microscope, Philips), Zetasizer Nano ZS (Malvern-Instruments), western-blot detection, and proteomic analysis by mass spectrometry were also carried out, as previously described [22 (link)]. Exosome quantification was performed using a Bicinchoninic acid kit (BCA; Thermo-Scientific).
6
Mitochondrial Ultrastructure Analysis in Astroglia
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Transmission electron microscopy (TEM) was used to study the fine structure of the mitochondria and to count the number of mitochondria, as described previously [60 (link)]. Briefly, astroglia derived from 3xTg mice were treated with/without nilotinib (100 nM) for 24 hours. After incubation, cells were washed with PBS and fixed 3 hours in 3% v/v glutaraldehyde in 0.1 M Sorensen’s buffer. Cells were again fixed in 1% osmium tetroxide in 0.1 M Sorensen’s phosphate buffer for two hours, and dehydrated in a 30% to 100% ethanol series and embedded with Epon resin and infiltrated for 4 days. Later, sections were polymerized at 60 °C for 30 hours; Images were collected with Morgagni 268D electron microscope (Philips, Netherlands).
7
Freeze Fracture Imaging of SSL
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A 1-2 μl droplet of SSLT or SSL suspension was used for freeze fracturing. The samples were pipetted to the golden sample holder and rapidly frozen in the mixture of liquid and solid Freon, cooled by liquid nitrogen. Fracturing was performed at 173 K in a freeze fracture device (BAF 400D, Balzers AG, Liechtenstein). The fractured surfaces were etched for 30 s at 173 K and then shadowed by platinum and covered with carbon. The replicas obtained were washed with surfactant and water and were finally transferred to 200-mesh copper grids. The electron micrographs were made in a Philips Morgagni 268D electron microscope.
8
Ultrastructural Analysis of Cardiac Tissue
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In order to perform TEM study, the heart samples conserved in formalin were retrieved. A 2 mm3 (link) large fragment was taken according to the supply regions of the damaged coronaries. After a thorough washing with phosphate buffer, the samples were post-fixed in glutaraldehyde for two hours and then immersed in a solution composed of osmium tetroxide (2%) and iron cyanide (3%) for 2 h. Then, after another thorough washing with phosphate buffer, specimens were dehydrated in increasing concentration alcohol solutions, and embedded in epon-araldite mixture. Semithin section were cut and stained with toluidine blue; after the observation at light microscopy, to make sure the fibers are longitudinally oriented, the ultrathin sections were cut at 70 nm thickness, placed on Cu/Rh grids, stained with lead citrate and observed with Morgagni 268D electron microscope (Philips).
9
Ultrastructural Analysis of Tissue
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Formalin-fixed, paraffin-embedded samples of brain tissue and genital mucosa were deparaffinized and embedded in epoxy resin. Thin sections were prepared, stained with 6% saturated uranyl acetate and lead citrate, and examined with a Philips Morgagni 268D electron microscope (F.E.I., Brno, Czech Republic).
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