Ultra microtome ultracut uct

The transmission electron microscopy images of hiPSC-derived AT2-like cells were generated following standard procedures. Shortly, 96-Transwell inserts were pre-fixed for 1 h with 2.5% glutaraldehyde (EM grade) and 2% Paraformaldehyde 0.1% cacodylate buffer solution. The PET membrane with pre-fixed cells was then carefully removed from the Transwell tray and washed with a 0.1% cacodylate buffer solution for 30 min. Subsequently, the samples were transferred into an EM-TP Tissue processor (Leica Biosystems, Nussloch, Germany) for automated post-fixation, staining, dehydration and embedding. The samples underwent the following preparation steps: 20 min in 0.1% cacodylate buffer, 3 h in 2% Daltons osmiumtetroxide aq., 3 times 15 min in 0.1% cacodylate buffer solution, 15 min in 30% isopropanol, 30 min each in 30%, 50%, 70%, 90% and 100% isopropanol, and three times 1 h in 100% isopropanol. Following dehydration, sample infiltration with Epoxy resin was achieved as follows: 30 min in 50% isopropanol/ 50% EPON, 30 min in 33% isopropanol/ 66% EPON, 30 min in 20% isopropanol/ 80% EPON and 60 min in 100% EPON. Afterwards, the samples were then incubated twice for 6 h in 100% EPON and hardened at 60 °C for 24 h. Ultra-thin sections (50 nm) were prepared on an Ultracut UCT ultra-microtome (Leica Biosystems, Nussloch, Germany) and imaged on a TEM 912AB (Zeiss, Oberkochen,Germany).

Cell pellets from broth culture or loopfuls of material from AMM plates were fixed for 48 h at 4 °C in 0.1 M cacodylate buffer (pH 7.4) containing glutaraldehyde/paraformaldehyde (2.5%/1%), then washed 3 times with cacodylate buffer. The pellets were dehydrated in ethanol and propylene oxide, then embedded in EMBed 812 resins (Electron Microscopy Sciences, Hatfield, PA, USA). Thin sections (80 nm) were cut using an Ultracut-UCT ultramicrotome (Leica Biosystems, Wetzlar, Germany), placed onto 300-mesh copper grids, then stained with saturated uranyl acetate in 50% methanol followed by lead citrate. Grids were viewed with a JEM-1200EXII electron microscope (JEOL Ltd., Tokyo, Japan) at 80 kV; images were recorded on a mid-mounted CCD camera (XR611M, 10.5 Mpixel; Advanced Microscopy Techniques Corp., Woburn, MA, USA).

For TEM analyses to evaluate cell morphologies and sub-organelle structures, drug-treated or control MDA-MB231 CD63-GFP fusion cells were pelleted by centrifugation, and pelleted cells were fixed with 2.5% glutaraldehyde in a 0.1 M phosphate buffer. After washing several times with 0.1 M carcodylate buffer, cells were dehydrated by gradient series of ethanol (50%, 60%, 70%, 80%, 90% ethanol for 20 min each step, 100% 20 min twice) followed by propylene oxide for twice. Afterwards, cells were infiltrated with progressively concentration Eponate 812, and then polymerized in fresh Eponate 812 for 2 days at 60 °C. Samples were sectioned using an Ultra microtome (Ultracut UCT; Leica) and stained with uranyl acetate and lead citrate. Sections were examined with energy filtering TEM (LEO-912AB OMEGA; Carl Zeiss) at the Korean Basic Science Institute Chuncheon Center.
For TEM analyses to detect sEV, MDA-MB231 and MCF7 cells were pelleted by serial ultracentrifugation, and the pellets were deposited on pure carbon-coated EM grids. After staining with 1% uranyl acetate, the grids were dried at room temperature and viewed at ×12,000 magnification using a Biotransmission electron microscope (HT7700; Hitachi) operated at 120 kV.