ISSN:
0741-0581
Keywords:
Quick-freeze deep-etch rotary replication
;
Cell monolayers
;
Tissue culture
;
Specimen preparation
;
Freeze-etch artifacts
;
Temperature control
;
Life and Medical Sciences
;
Cell & Developmental Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Natural Sciences in General
Notes:
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 (〈 10 μm). 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.
Additional Material:
3 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/jemt.1060140409
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