Ultrascan 1000 digital camera

HEK293T cells (Life Technologies) were transfected with Usp12-FUGW2 or GFP-FUGW, and delta8.9 and VSVG plasmids to generate lentivirus. Supernatant was collected 48–96 h after transfection and concentrated by PEG precipitation and centrifugation. Lentivirus was titered with Lenti-X p24 Rapid Titer Kit (Clontech). Primary rodent neuronal cultures transduced with GFP or Usp12 lentivirus for approximately 5 days, were fixed in 2% glutaraldehyde, 1% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, post-fixed in 2% osmium tetroxide in the same buffer, en block stained with 2% aqueous uranyl acetate, dehydrated in acetone, infiltrated, and embedded in LX-112 resin (Ladd Research Industries). Samples were ultrathin-sectioned on a Reichert Ultracut S ultramicrotome and counterstained with 0.8% lead citrate. Grids were examined on a JEOL JEM-1230 transmission electron microscope (JEOL USA, Peabody, MA) and photographed with the Gatan Ultrascan 1000 digital camera (Gatan, Warrendale, PA).

MCF-10A and MCF-7 cells were cultured in 10-cm plates in preparation for transmission electron microscopy (TEM) analysis. Following the completion of treatment with 10 mL of either 30 μM C-6 or a matched DMSO vehicle control, the cells were fixed in a pH 7.4 0.1 M sodium cacodylate buffer containing 2.5% glutaraldehyde, 1% paraformaldehyde, 2.4% sucrose, and 8 mM calcium chloride. After fixation, the cells were rinsed in 0.1 M sodium cacodylate buffer and post-fixed in 2% osmium tetroxide also in the 0.1 M sodium cacodylate buffer. Cells were rinsed in type 1 water and enbloc stained in saturated aqueous uranyl acetate. Cells were then dehydrated in a graded ethanol series, transitioned through acetone, infiltrated with Embed 812 and acetone, embedded in fresh Embed 812, and allowed to cure overnight in a 60°C oven. Plastic sections were cut on a Leica (Wetzlar, Germany) ultramicrotome with a diamond knife and placed on copper grids at a thickness of 80 to 100 nm. Sections were contrasted with saturated aqueous uranyl acetate followed by Reynold’s lead citrate. They were then examined on an FEI Tecnai (Hillsboro, OR, USA) T-12 TEM with a LaB6 filament at 120KV. Images were acquired with a Gatan (Pleasanton, CA, USA) Ultrascan 1000 digital camera using Gatan’s digital micrograph.

Cells were collected at 48 hours post infection and fixed in 2% glutaraldehyde, 1% paraformaldehyde in 0.1 M sodium cacodylate buffer pH 7.4, followed by post-fixation in 2% osmium tetroxide in the same buffer, staining with 2% aqueous uranyl acetate, dehydration in acetone, and embedding in LX-112 resin (Ladd Research Industries, Burlington, VT). Samples were sectioned on a Reichert Ultracut S ultramicrotome and counterstained with 0.8% lead citrate. Grids were examined on a JEOL JEM-1230 transmission electron microscope (JEOL USA, Inc., Peabody, MA) and photographed with the Gatan Ultrascan 1000 digital camera (Gatan Inc., Warrendale, PA).