Jem 1400 electron microscope

EVs were purified from coACCM and AF using the ExoQuick TC-ULTRA kit (System Bio, Palo Alto, CA, USA) or AF EVs were purified by ultracentrifugation with a Beckman XL-80 Ultracentrifuge (Beckman Coulter, Brea, CA, USA) and a SW41TI rotor by 107,000× g spin for 90 min at 4 °C. The resulting supernatant was collected as the EV-depleted fraction, and the pellet was washed twice with cold PBS, then resuspended in cold PBS and immediately stored at −80 °C or on ice then further analyzed. Qualitative and quantitative analysis of EVs was performed using nanoparticle tracking analysis (NTA) with the ZetaView particle analyzer model PMX-120 (Particle Metrix, Inning am Ammersee, Germany), transmission electron microscopy (see below), the Bradford method as described previously [72 (link)] (BioRad, Hercules, CA, USA), Western blotting (see below), and LC-MS/MS (see below).
For TEM either total AF, purified EVs from AF, or EV-depleted AF were applied to electron microscopic 200 mesh copper grids and negatively stained with 2% aqueous uranyl acetate. They were examined and measured in a JEOL JEM-1400 electron microscope at 80 kV. Images were acquired on bottom-mounted CCD camera Orius SC200 (Gatan, Pleasanton, CA, USA), and were used to measure the size, relative numbers, and shape of EVs.

Tissues were fixed in 4% paraformaldehyde and then paraffin. Sections were cut into 5 μm sections using a microtome. Tissues were stained with hematoxylin and eosin (H&E) and examined using light microscopy. Fresh corneal and conjunctival samples were fixed in 2.5% glutaraldehyde solution in phosphate buffer at pH 7.4 for 4 h and postfixed for 1 h in 1% osmium tetroxide solution in 0.1 M phosphate buffer solution. After further postfixation, dehydration, embedding, slicing, and staining procedures, sections were examined in JEOL JEM-1400 electron microscope and photographed by CCD camera (Gatan Inc., Pleasanton, CA, USA).