Tecnai transmission electron microscope

VLPs and viruses were analyzed by electron microscopy as described [67 (link)]. For cell binding assays, 5 μl of KSHV glycoprotein-based purified VLPs (gpK8.1, gB, or gH/gL) were incubated with HMC-1 cells for 10 min at room temperature and detected with anti-gpK8.1 mAb, polyclonal anti-gB, or polyclonal anti-gH. Lipid rafts in the cell membranes were stained with cholera toxin (cell membrane tracker) and nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). For electron microscopy, purified VLPs were dialyzed against 1 L of TNE buffer (100 mM Tris; 2.0 M NaCl; 10 mM EDTA; pH 7.4) to remove residual sucrose, incubated with 3% bovine serum albumin (BSA) in TNE for 45 min, and embedded on a grid. 5 μl of the virus/VLP at 1:40 dilution was individually added to the grid for 1 h at room temperature. After two final washes, the grids were negatively stained with 12% phosphotungstic acid (pH 7) for 15 sec, air dried for 30 min, and examined using a Tecnai transmission electron microscope (FEI).

The lens capsule specimens from cataract patients were immediately fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (Cat.# G5882, Sigma) for 2 h at RT. After thorough rinsing in 0.1 M phosphoric acid solution, the specimens were post-fixed with 1% osmium tetroxide (Cat.# 365092, Sigma) for 1 h and rinsed three times in water. The specimens were then stained with 2% uranyl acetate for 0.5 h at 4 °C before dehydration through a graded ethanol series. Embedding agent and pure acetone (1:1, 3:1) were added for gradient embedding, and specimens were finally mixed with pure embedding agent and incubated at 37 °C. The specimens were cut using an ultra-thin trimming machine and the ultrastructural features of autophagy were observed and imaged under a Tecnai transmission electron microscope (FEI, USA) at an operating voltage of 80 kV.

Expression of UAS-fluorescent protein transgenes (cytosolic GFP, mCherry-Atg8a, GFP-Atg8a, and mRFP-Rab4) was controlled by a Gal4 driver and fluorescence detected using confocal microscopy on adult brains fixed in PBS with 4% formaldehyde for 30 min, followed by thorough washing in PBS. Electron microscopy was performed as previously described (Ling et al., 2009 (link)) using an FEI Tecnai transmission electron microscope. Independent observations from three to five animals were performed for each experimental condition.

Transmission electron microscope analysis was performed on the gastrocnemius in standard fashion. Ultra‐thin sections were stained with uranyl acetate and lead citrate, and then examined using a Tecnai transmission electron microscope (FEI) operated at 80 kV.

Cells were grown in MEM or DMEM, washed twice with PBS and fixed for 5 min in Karnovsky fixative (2.5% glutaraldehyde, 2.5% paraformaldehyde in cacodylate buffer pH 7.4) on the tissue culture plate. Cells were then collected, and transferred to 1.5 ml tube for further 1 h fixation at room temperature. The cells were then stored O.N at 4 °C after which the samples were washed in 0.1 M cacodylate buffer and post-fixed with 1% OsO4 in 0.1 M sodium cacodylate buffer (Sigma-Aldrich, Israel) for 1 h. Samples were then dehydrated in alcohol and propylene oxide followed by embedding in Agar Mix. Thin sections (60 nm) were cut, stained with uranyl acetate and lead citrate, and observed in a FEI Tecnai transmission electron microscope.

Cells were grown on Thermanox coverslips (Nalgene, Nunc) and fixed with 2% PFA, 2.5% glutaraldehyde in 0.1 M cacodylate. Cells were then secondarily fixed with 1% osmium tetroxide followed by incubation with 1% tannic acid to enhance contrast. Cells were dehydrated using increasing percentages of ethanol before being embedded onto Epoxy resin (Agar scientific, UK) stubs. Coverslips were cured overnight at 65 °C. Ultrathin sections were cut using a diamond knife mounted to a Reichert ultracut S ultramicrotome and sections were collected onto copper grids. Grids were post-stained with drops of lead citrate. Sections were viewed on a FEI Tecnai transmission electron microscope (Eindhoven, The Netherlands) at a working voltage of 80 kV. BSA-gold was prepared as previously described [27 (link)]. For quantification of MVB diameter, MVBs were defined as organelles containing intraluminal vesicles and monomeric rather than flocculated BSA-gold.
Aβ1-42 peptides (Sigma) were incubated at 37 °C for 1 h to induce fibril formation in vitro. Grids were inverted onto the drops of Aβ1-42, negatively stained with 2% uranyl acetate, washed with water and dried on filter paper before being viewed by EM.