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Lab-Tek Chamber Slide for TEM Prep: A Simple, Rapid, and Reliable Protocol for In Situ Embedding Monolayer Cell Cultures in Epoxy and LR White Resin

Published online by Cambridge University Press:  14 March 2018

Gang Ning*
Affiliation:
Penn State University, State College, PA

Extract

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Preparation for TEM of resin-embedded monolayer cell cultures usually requires scraping cells from the culture substrate before or after primary fixation, which disturbs the monolayer and often results in mechanical trauma to the cells. After harvest, the cells are centrifuged and processed as a pellet in a microcentrifuge tube through a prolonged procedure of post-fixation, dehydration, infiltration, and finally embedding to ensure a “well done” sample block for subsequent sectioning, staining, and observing under a transmission electron microscope. Other disadvantages to this method include the loss of cellular orientation and information on cellular interaction, as well as difficulty in collecting a large quantity of cells to form a sizable pellet for processing, which is of special importance when samples are differentiated cells and neurons cultured in low density. In an alternative method, cells can be seeded on either glass or plastic cover slips or Petri dishes, and then processed and embedded directly as a monolayer.

Type
Research Article
Copyright
Copyright © Microscopy Society of America 2008

References

Harb, J.M. (1993), Interpretation of TEM Micrographs for Human Diagnosis. MSA Bulletin 23 (4) 206-218. The formula of the epoxy resin is 76g Eponate 12, 18g Araldite 502, 39g DDSA, 61g NMA, and 2-3.5% DMP-30.Google Scholar
Bozzola, J.J. (2006), Conventional Specimen Preparation Techniques for Transmission Electron Microscopy of Cultured Cells, in Electron Microscopy: Methods and Protocols, 2nd Ed (Kuo, J. ed.), Methods in Molecular Biology, vol. 369.Google Scholar