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Journal of Histochemistry and Cytochemistry, Vol. 45, 595-598, Copyright © 1997 by The Histochemical Society, Inc.

TECHNICAL NOTE

Metal Sandwich Method to Quick-freeze Monolayer Cultured Cells for Freeze-fracture

Toyoshi Fujimotoa and Kazushi Fujimotob
a Department of Anatomy and Cell Biology, Gunma University School of Medicine, Maebashi, Japan
b Department of Anatomy, Faculty of Medicine, Kyoto University, Kyoto, Japan

Correspondence to: Toyoshi Fujimoto, Dept. of Anatomy and Cell Biology, Gunma Univ. School of Medicine, 3-39-22 Showa-machi, Maebashi 371, Japan.

We describe a simple quick-freezing method to obtain a large fractured plane of the plasma membrane from monolayer cultured cells. Cells were grown on thin gold foil, inverted on a thin layer of gelatin on thin copper foil, and frozen by a quick press between two gold-plated copper blocks precooled in liquid nitrogen. The frozen cell sandwich was mounted on the cold stage of a freeze-fracture device with the gold side up and was fractured by separating the sandwich with a cold fracture knife. When this technique was applied to confluent monolayer cells, large replicas of the E-face of the upper plasma membrane and the P-face of the lower plasma membrane were obtained. The present metal sandwich method is simple, does not require any expensive equipment, and provides a large fracture plane of the plasma membrane for subsequent histochemical manipulation. (J Histochem Cytochem 45:595-598, 1997)

Key Words: quick freezing, freeze-fracture, freeze replica, plasma membrane, cultured cells, caveolin, immunocytochemistry


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