Quick-freeze, deep-etch replication of cells in monolayers. 1990

D W Pumplin, and P W Luther, and S J Samuelsson, and J A Ursitti, and J Strong
Department of Anatomy, University of Maryland School of Medicine, Baltimore 21201.

We have made several technical improvements for quick-freeze, deep-etch replication of monolayers of cells grown on, or attached to, glass coverslips. Cells studied include muscle cells of rat and Xenopus cultured on glass coverslips, and erythrocytes attached to coverslips coated with poly-L-lysine. We describe methods for identifying particular areas of cultures, e.g., clusters of acetylcholine receptors on muscle cells, by light microscopy and then relocating these areas after replication. For good preservation of structure by quick-freezing, it is necessary to ensure that the surface to be frozen is covered by a minimum depth of water (less than 10 microns). Insufficient or excess water left on the sample during freezing causes recognizable artifacts in its replica. We describe two ways to control the water table--one by improving visual control of water removal, the other by blowing excess water off the sample surface with a jet of nitrogen applied during its descent to the freezing block. Finally, we describe a new specimen holder that allows us to etch and replicate six samples at once with good thermal contact between the stage and samples.

UI MeSH Term Description Entries
D009132 Muscles Contractile tissue that produces movement in animals. Muscle Tissue,Muscle,Muscle Tissues,Tissue, Muscle,Tissues, Muscle
D002478 Cells, Cultured Cells propagated in vitro in special media conducive to their growth. Cultured cells are used to study developmental, morphologic, metabolic, physiologic, and genetic processes, among others. Cultured Cells,Cell, Cultured,Cultured Cell
D005614 Freeze Fracturing Preparation for electron microscopy of minute replicas of exposed surfaces of the cell which have been ruptured in the frozen state. The specimen is frozen, then cleaved under high vacuum at the same temperature. The exposed surface is shadowed with carbon and platinum and coated with carbon to obtain a carbon replica. Fracturing, Freeze,Fracturings, Freeze,Freeze Fracturings
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D051381 Rats The common name for the genus Rattus. Rattus,Rats, Laboratory,Rats, Norway,Rattus norvegicus,Laboratory Rat,Laboratory Rats,Norway Rat,Norway Rats,Rat,Rat, Laboratory,Rat, Norway,norvegicus, Rattus

Related Publications

D W Pumplin, and P W Luther, and S J Samuelsson, and J A Ursitti, and J Strong
January 1989, Journal of electron microscopy,
D W Pumplin, and P W Luther, and S J Samuelsson, and J A Ursitti, and J Strong
July 1984, Journal of cell science,
D W Pumplin, and P W Luther, and S J Samuelsson, and J A Ursitti, and J Strong
January 1986, Methods in enzymology,
D W Pumplin, and P W Luther, and S J Samuelsson, and J A Ursitti, and J Strong
January 1981, Methods in cell biology,
D W Pumplin, and P W Luther, and S J Samuelsson, and J A Ursitti, and J Strong
May 1994, Microscopy research and technique,
D W Pumplin, and P W Luther, and S J Samuelsson, and J A Ursitti, and J Strong
August 1995, Nippon Ganka Gakkai zasshi,
D W Pumplin, and P W Luther, and S J Samuelsson, and J A Ursitti, and J Strong
February 1985, The Journal of cell biology,
D W Pumplin, and P W Luther, and S J Samuelsson, and J A Ursitti, and J Strong
May 1990, The Anatomical record,
D W Pumplin, and P W Luther, and S J Samuelsson, and J A Ursitti, and J Strong
January 1989, Journal of electron microscopy,
D W Pumplin, and P W Luther, and S J Samuelsson, and J A Ursitti, and J Strong
January 1991, Cell and tissue research,
Copied contents to your clipboard!