Human embryonic lung fibroblast–derived WI-38 cells were imaged by conventional TEM as described earlier (44 (link)). Briefly, cells were grown on glass coverslips, fixed with 2.5% glutaraldehyde and 2% paraformaldehyde (in 0.1 M sodium cacodylate buffer at 25°C), washed in cacodylate buffer (4°C), and stained with 1% osmium tetroxide and 2% uranyl acetate. Samples were dehydrated in cold ethanol and embedded in Epon. Upon removal of the glass coverslips with liquid nitrogen, ultrathin sections (~70 nm) were cut parallel to the coverslip surface and transferred to 200 mesh copper grids. Sections were imaged in a 120 kV FEI Tecnai Spirit T-12 equipped with a 2k Eagle CCD camera (Thermo Fisher Scientific, FEI).
Carter S.D., Hampton C.M., Langlois R., Melero R., Farino Z.J., Calderon M.J., Li W., Wallace C.T., Tran N.H., Grassucci R.A., Siegmund S.E., Pemberton J., Morgenstern T.J., Eisenman L., Aguilar J.I., Greenberg N.L., Levy E.S., Yi E., Mitchell W.G., Rice W.J., Wigge C., Pilli J., George E.W., Aslanoglou D., Courel M., Freyberg R.J., Javitch J.A., Wills Z.P., Area-Gomez E., Shiva S., Bartolini F., Volchuk A., Murray S.A., Aridor M., Fish K.N., Walter P., Balla T., Fass D., Wolf S.G., Watkins S.C., Carazo J.M., Jensen G.J., Frank J, & Freyberg Z. (2020). Ribosome-associated vesicles: A dynamic subcompartment of the endoplasmic reticulum in secretory cells. Science Advances, 6(14), eaay9572.
Corresponding Organization : University of Pittsburgh
Other organizations :
Centro Nacional de Biotecnología, University of California, San Francisco, Columbia University Irving Medical Center, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, New York Psychoanalytic Society and Institute, New York Structural Biology Center, Institut de Biologie Paris-Seine, Centre National de la Recherche Scientifique, Hospital for Sick Children, Weizmann Institute of Science
Ultrastructural morphology of human embryonic lung fibroblast–derived WI-38 cells
control variables
Growth of WI-38 cells on glass coverslips
Fixation with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer at 25°C
Washing in cacodylate buffer at 4°C
Staining with 1% osmium tetroxide and 2% uranyl acetate
Dehydration in cold ethanol
Embedding in Epon
Removal of glass coverslips with liquid nitrogen
Cutting of ultrathin sections (~70 nm) parallel to the coverslip surface
Transferring ultrathin sections to 200 mesh copper grids
Imaging in a 120 kV FEI Tecnai Spirit T-12 equipped with a 2k Eagle CCD camera
controls
None explicitly mentioned
Annotations
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