Ultracut 6 ultramicrotome

Spleens from Sec13^+/+ and Sec13^H/− mice were cut into 1 mm³ pieces and fixed with 2.5% (v/v) glutaraldehyde in 0.1 M sodium cacodylate buffer. Tissues were then rinsed in 0.1 M sodium cacodylate buffer and post-fixed in 1% osmium tetroxide and 0.8% Potassium Ferricyanide in 0.1 M sodium cacodylate buffer for 1.5 h at room temperature. Tissues were rinsed with water and en bloc stained with 4% uranyl acetate in 50% ethanol for 2 h. Next, tissues were dehydrated with increasing concentrations of ethanol, transitioned into resin with 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 6 ultramicrotome (Leica Microsystems) and collected onto copper grids, post-stained with 2% aqueous Uranyl acetate and lead citrate. Images were acquired on a Tecnai G² spirit transmission electron microscope (FEI) equipped with a LaB₆ source using a voltage of 120 kV.

Hs578T cells expressing three different shNAs targeting NDRG1 and one targeting enhanced (e)GFP were collected at one week post selection. Cells were grown in 10-cm dishes to 80–90% confluence and fixed in 0.1 M cacodylate pH 7.4, 2.5% glutaraldehyde (Electron Microscopy Sciences). Cells were fixed at 37 °C for 15 min, followed by scraping of cells, collection by centrifugation at × 100 g, and resuspension in 10 ml fresh fixation buffer and storage at 4 °C. After three rinses with 0.1 M sodium cacodylate buffer, cell pellets 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. Cells were rinsed with water and stained en bloc with 4% uranyl acetate in 50% ethanol for 2 h. Cells were dehydrated with increasing concentration of ethanol, transitioned into propylene oxide, infiltrated with Embed-812 resin and polymerized in an oven at 60 °C overnight. Blocks were sectioned with a diamond knife (Diatome) on a Leica Ultracut 6 ultramicrotome (Leica Microsystems) and collected onto copper grids, post-stained with 2% aqueous uranyl acetate and lead citrate. Images were acquired on a Tecnai G2 spirit transmission electron microscope (FEI) equipped with a LaB6 source using a voltage of 120 kV.