Tecnai g2 electron microscope

After anesthetization using pentobarbital, the eyes were harvested, placed on ice, and fixed with Karnovsky's solution for transmission electron microscope observation. The tissues were cut into small pieces, stored in 2% glutaraldehyde, fixed with osmium tetroxide, and embedded in an Epon 812 mixture. Samples were sectioned using an ultramicrotome, stained with uranyl acetate and lead citrate, and examined under an FEI Tecnai G2 electron microscope (FEI, Eindhoven, The Netherlands).

Ribbons of serial thin sections (200 nm thick) were collected on formvar coated slot grids, then post-stained with aqueous uranyl acetate and Renyold’s lead citrate. Colloidal gold particles were deposited on both surfaces of the sections for use as fiducial markers during subsequent image alignment. Sections were viewed in an FEI Tecnai-G2 electron microscope operating at 200 KeV, and images recorded digitally with a FEI Eagle 4K bottom mount CCD camera (FEI Tecnai, OR) using a pixel size of 1.2 nm. Tilt series were recorded with automated methods for image montaging, data acquisition and image alignment as the sample was serially tilted along single axis from at −65° to +65°, by 1° angular increments over a range between +40° and −40°, and by 2° angular increments over the rest. 3D distributions of stain density (tomograms) were calculated from each tilt series on FEI Xpress3D reconstruction (FEI Tecnai, OR). Tomographic reconstructions were aligned with each other and combined to produce a single dual-axis 3D reconstruction on Amira v.5.4.0 software. Tomograms from adjacent sections were aligned to each other, then subcellular structures and membranes within the 3D volumes were analyzed and modeled.

Infected HeLa cells were washed with PBS (3×) and cooled on ice before fixation with 0.5% glutaraldehyde (Agar Scientific) in 200 mM sodium cacodylate (TAAB) for 5 min on ice, then at RT for a further 25 min. Cells were immediately washed in cacodylate buffer and Tf-HRP reacted with diaminobenzidine (DAB) in stable peroxide buffer (Metal Enhanced DAB Substrate Kit, Thermo Scientific) for 30 min at RT. The reaction was stopped by washing in sodium cacodylate before post-fixation in 1% osmium tetroxide/1.5% potassium ferrocyanide for 1 h at RT. The cells were then washed in ddH₂O, stained overnight at 4°C with 0.5% uranyl acetate, washed with ddH₂O and serially dehydrated in graded ethanol before infiltration with Epon 812 resin. Ultrathin sections (∼70 nm) of the flat-embedded cell monolayers were cut parallel to the surface of the dish, collected onto formvar-coated 50 mesh EM grids, and stained for 30 s with Reynolds' lead citrate before imaging. TEM samples were viewed by using an FEI Tecnai G² electron microscope with a Soft Imaging System Megaview III charged-coupled-device camera. Images were collected at 1376 by 1032 by 16 pixels using AnalySIS version Docu software (Olympus Soft Imaging Solutions).

S2 cells were fixed in 4% paraformaldehyde, cryoprotected, frozen by immersion in liquid nitrogen and cryosectioned. The primary antibodies were anti-RRP6 antibody [20 (link)] and anti-HP1a antibody. The secondary antibodies were conjugated to 6 nm and 12 nm gold particles (Jackson ImmunoResearch Laboratories). After immunolabelling, the sections were stained with 2% aqueous uranyl acetate, embedded in polyvinyl alcohol and examined in a FEI Tecnai G2 electron microscope at 80 kV.

For energy-dispersive X-ray spectroscopy (EDX) analyses, ultrathin sections and freeze fracture replicas were measured using a Tecnai G2 electron microscope (FEI, Eindhoven, Netherlands). High-angle annular dark-field (HAADF) images were acquired at 200 kV, and the electron beam was operated in STEM (scanning transmission electron microscopy) mode. For the detection of lanthanum, a multipoint EDX analysis of the samples was performed by using an energy-dispersive X-ray spectrometer system, Quantax 200, with an XFlash detector (model 5030; Bruker, Berlin, Germany).