0.1 mol l sodium cacodylate buffer

Electron microscopy (EM) was performed in the Microscopy Core in the Program for Membrane Biology at Massachusetts General Hospital. Intestinal tissues were fixed in 2.0% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer (Electron Microscopy Sciences, Hatfield, PA) overnight at 4°C. The following day, samples were postfixed in 1.0% osmium tetroxide for 1 hour at room temperature, rinsed in buffer, and dehydrated through a graded series of ethanol. Samples then were infiltrated with Epon resin (Ted Pella, Redding, CA) in a 1:1 solution of Epon:ethanol overnight. The following day, they were placed in fresh Epon for several hours and then embedded in Epon overnight at 60°C. Thin sections were cut on a Leica (Wetzlar, Germany) EM UC7 ultramicrotome, collected on formvar-coated grids, stained with uranyl acetate and lead citrate, and examined in a JEOL (Peabody, MA) JEM 1011 transmission electron microscope at 80 kV. Images were collected at direct magnification of 30,000× using an AMT digital imaging system (Advanced Microscopy Techniques, Danvers, MA).

Heart tissue was fixed overnight in 2.5% Glutaraldehyde in 0.1 mol/L sodium cacodylate buffer (Electron Microscopy Sciences) as previously described in our laboratory.⁵ (link)^,6 (link) After postfixation with 1% osmium tetroxide in 0.1 mol/L cacodylate buffer, the tissue was dehydrated with a graded series of ethanol and embedded in Epon resin. Semi-thin and ultra-thin sections were cut, mounted on copper grids, and poststained with uranyl acetate and lead citrate. Sections were viewed in a transmission electron microscope for qualitative changes in mitochondria and cardiomyocyte ultrastructure.⁵ (link)^,6 (link) To supplement the human studies, mechanistic studies were performed in a canine model of PMR.² (link)^,3 (link) The methods and results of these studies are presented in the Supplemental Material.

LSPs and CPPs were fixed in 4% PFA for 30 min, cytospun onto glass slide, washed with PBS, fixed with 2% glutaraldehyde (GTA) for 1 h, washed extensively with 0.1 mol/L sodium cacodylate buffer (Electron Microscopy Sciences, Hatfield, PA), dehydrated in ethanol series and dried at RT, sputter coated using a high-resolution sputter coater (Ted Pella, Inc., Redding, CA) with a thin film of gold at 13.3 Pa and 45 mA for 90 s. The images were collected with Hitachi S4700 microscope (28 (link)). To estimate damaged PLTs in CPPs, 7 large-field images were collected, and percentages of damaged PLTs with rough, porous membranes and grape-like structures were counted.