Cm120 biotwin transmission electron microscope

TEM analysis of the MTs at the fat body ncMTOC was performed essentially as described^{67 (link)}, except that fat bodies from 30 third instar larve were fixed for 72 hrs in 1 ml Karnovsky’s fixative (EM Sciences Cat#15720). Embedding, staining, sectioning and preparation of grids were performed by the Core Facility at UT Southwestern Medical Center. After three rinses with 0.1 M sodium cacodylate buffer, samples were embedded in 3% agarose and sliced into small blocks (1mm³), rinsed with the same buffer three times and post-fixed with 1% osmium tetroxide and 0.8 % potassium ferricyanide in 0.1 M sodium cacodylate buffer for 1.5 h at room temperature. Samples were rinsed with water and en bloc stained with 4% uranyl acetate in 50% ethanol for 2 h. They were then dehydrated with increasing concentration of ethanol, transitioned into propylene oxide, infiltrated with Embed-812 resin and polymerized in a 60°C oven overnight. Blocks were sectioned with a diamond knife (Diatome) on a Leica Ultracut 7 ultramicrotome (Leica Microsystems) and collected onto copper grids, post stained with 2% aqueous Uranyl acetate and lead citrate. Images were acquired on a Phillips CM120 Biotwin transmission electron microscope at the Florida State University Biological Science Imaging Resource (BSIR).