PMID: 7012364Feb 1, 1981Paper

Cryofixation of monolayer cell cultures for freeze-fracturing without chemical pre-treatments

Journal of Microscopy
P PscheidH Plattner

Abstract

A cryofixation method is presented which gives excellent ultrastructural preservation of monolayer cell cultures without any chemical pretreatments. Rat hepatocytes in primary culture were used in this study. The equipment needed is inexpensive and easy to manufacture. Cells are grown on a usual tissue culture support material (Thermanox plastic sheets). For cryofixation, samples are prepared essentially by a combined sandwich-cryogen-jet technique, 3 mm large discs are punched out and sandwiched with Cu- or Au-object holders of little mass; a 15 micrometer spacer is put in between. The viability of the cells is not impaired by the manipulations before freezing. The sandwich sample is quickly frozen by shooting a propane jet from a simple pressure chamber on to the metal object holder. The relevant parameters were optimized by parallel freeze-fracture analyses of 5% glycerol as a model system and by thermocouple measurements. Sandwich samples are then mounted in an appropriate double replication specimen table for further analysis by freeze-fracturing. It is possible to obtain a certain selectivity of the fracture plane with regard to apical, lateral or basal aspects of the cell layer. Alternatively, disc samples can be process...Continue Reading

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Citations

Feb 9, 1983·Biochimica Et Biophysica Acta·J A KillianJ de Gier
Sep 30, 1991·Biochimica Et Biophysica Acta·K N BurgerA J Verkleij
Sep 1, 1991·Journal of Microscopy·S M Bailey, J A Zasadzinski
Jan 1, 1982·Ultrastructural Pathology·G M RoomansL Sevéus
Apr 1, 1982·Journal of Microscopy·H Y ElderW H Biddlecombe
Oct 1, 1985·Journal of Microscopy·K P Ryan, D H Purse
Dec 1, 1989·Journal of Electron Microscopy Technique·J A Zasadzinski, S M Bailey

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