Em pact2 high pressure freezer

We used samples that were previously obtained for a 3D reconstruction of the first mitotic spindle in C. elegans^{46 (link)}. Briefly, C. elegans N2 (wild type) gravid adults were dissected in M9 buffer, and zygotes in early mitosis were collected in cellulose capillary tubes (Cat# 16706869, Leica Microsystems) (Pelletier et al., 2006). The embryos were observed under a stereoscope until metaphase was reached and then immediately frozen using an EMPACT2 high-pressure freezer equipped with a rapid transfer system (RTS, Leica Microsystems). Freeze substitution of the cryo-immobilized embryos was done over three days in anhydrous acetone containing 1% OsO4 and 0.1% UA using freeze-substitution equipment (EM AFS, Leica Microsystems, (Pelletier et al., 2006)). Epon/Araldite infiltration was followed by thin-layer embedding and polymerization for three days at 60°C. After remounting the specimens on dummy blocks, serial semi-thick sections (300 nm) were cut using an ultramicrotome (Ultracut UCT, Leica Microsystems). Sections were collected on a Formvar-coated copper slot grids (EMS) and post-stained with 2% UA (in 70% methanol) followed by brief exposure to Reynold’s lead citrate Error! Hyperlink reference not valid..

Following final fixation in 2.5% glutaraldehyde, platelets were high pressure frozen using a Leica EM PACT2 high pressure freezer with rapid transfer system. Samples were transferred under liquid nitrogen to cryovials containing 2% osmium tetroxide/0.1% glutaralde-hyde/1%H2O in acetone. Samples were freeze substituted in a Leica EM AFS2 freeze substitution and low temperature embedding system under the following schedule: −90 ° C for 40 h, warm 3°C/h to −60°C, −60°C for 8 h, warm 3°C/h to −30°C, −30°C for 8 h, warm 3°C/h to 0°C. Following freeze substitution, samples were rinsed three times in acetone followed by tannic acid at 4°C (1% tannic acid with 1% H2O in acetone) for 1 h. Samples were rinsed three times in acetone followed by 1 h osmium wash (1% osmium tetroxide/1% H2O solution in acetone) at 4°C. Samples were rinsed 3x in acetone and embedded in resin starting at a 25% concentration.

HeLa cells were grown in a 6-well plate and siRNA treated for 65 h, trypsinized and pelleted for 1 min at 1,000g in cell culture media containing 5% FBS and 20% BSA. Cells were then immediately cryofixed using a Leica EM PACT2 high pressure freezer and then further processed as described34 , except that tannic acid was omitted from the freeze substitution medium. Blocks were sectioned on a Leica UC7 ultramicrotome using a diamond knife (Diamtome). 90-nm sections were transferred to 200-mesh Nickel grids and then immunolabelled as follows: grids were floated section side down on a 20 μl droplet of blocking solution (10% goat serum in TBS) for 15 min, then blotted and incubated on a droplet of primary (J2) antibody (diluted 1:25 in buffer A (1% BSA, 1% goat serum, 0.01% Tween-20 in TBS)) for 2 h at room temperature. Grids were washed by passing them over five droplets of buffer A, 5 min each, then incubated with secondary antibody (ab27242, goat anti-mouse conjugated to 20 nm gold) diluted 1:10 in buffer A for 90 min at room temperature, then washed by passing over three droplets of water. Sections were then post-stained for 10 min with uranyl acetate and Reynold’s lead citrate and imaged on a Tecnai 12 transmission electron microscope (FEI) equipped with a Gatan OneView CMOS camera.