Formvar silicon monoxide coated 200 mesh copper grids

EVs were imaged by the Electron Microscopy Core Facility at the University of Nebraska Medical Center. Briefly, samples for TEM imaging suspended in HEPES buffered saline were spotted onto formvar/silicon monoxide-coated 200 mesh copper grids (Ted Pella Inc., Redding, CA). Grids were glow discharged for 60 s at 20 µA prior to use. Samples were negatively stained with NanoVan (Nanoprobes, New York, NY) and examined on a FEI Tecnai G² Spirit TWIN transmission electron microscope (Hillsboro, OR) operating at an accelerating voltage of 80 kV. Images were acquired digitally with an AMT digital imaging system (Woburn, MA). Representative images including diameter measurements are shown in Fig. 1B.

The hypoxic and normoxic swine tenocytes were trypsinized, and centrifuged at 350 g for 5 min to form the pellet. The pellet was then fixed overnight using 2% glutaraldehyde in 0.1 M Sorenson’s buffer (pH 7.4) at 4 °C and ultra-thin sections of the cell pellets were made using ultra-microtome for TEM analysis following the previously published protocols^{47 ,48 (link)}. The sections for TEM imaging were spotted onto formvar/silicon monoxide coated 200 mesh copper grids (Ted Pella Inc. Redding, CA). Grids were glow discharged for 60 seconds at 20 µA with a GloQube glow discharge unit (Quorum Technologies, East Sussex, UK) prior to use. The sections were negatively stained with NanoVan (Nanoprobes, New York, NY) and examined on a Tecnai G² Spirit TWIN (FEI, Hillsboro, OR) operating at an accelerating voltage of 80 kV. Images were acquired digitally with an AMT (Woburn, MA) digital imaging system. The images were assessed for the morphological features such as paracrystalline inclusions, cristae organization, concentric layering of cristae membrane, matrix compartmentalization, and nano-tunneling to examine the mitochondrial pathology⁴⁷ . The aspect ratio (ratio of length to width) was calculated from the linear axis and cross-sectional axis of mitochondria using ImageJ software. The incomplete images of mitochondria were omitted from the analysis.