[논문]Rapid freeze-substitution preserves membranes in high-pressure frozen tissue culture cells

HAWES, P.; NETHERTON, C. L.; MUELLER, M.; WILEMAN, T.; MONAGHAN, P.

doi:10.1111/j.1365-2818.2007.01767.x

Rapid freeze-substitution preserves membranes in high-pressure frozen tissue culture cells

Journal of microscopy, v.226 no.2, 2007년, pp.182 - 189

HAWES, P. (Institute for Animal Health, Ash Road, Pirbright, Woking, Surrey GU24 0NF, U.K.) , NETHERTON, C. L. (Institute for Animal Health, Ash Road, Pirbright, Woking, Surrey GU24 0NF, U.K.) , MUELLER, M. (Domaine du Parc St. Georges, F-30200 Venejan, France) , WILEMAN, T. (School of Medicine, Health Policy and Practice, University of East Anglia, Norwich NR4 7TJ, U.K.) , MONAGHAN, P. (Institute for Animal Health, Ash Road, Pirbright, Woking, Surrey GU24 0NF, U.K.)

Abstract ▼ AI-Helper

SummaryWe describe a method for high-pressure freezing and rapid freeze-substitution of cells in tissue culture which provides excellent preservation of membrane detail with negligible ice segregation artefacts. Cells grown on sapphire discs were placed ‘face to face’ without removal of tissue culture medium and frozen without the protection of aluminium planchettes. This reduction in thermal load of the sample\\/holder combination resulted in freezing of cells without visible ice-crystal artefact. Freeze-substitution at −90°C for 60 min in acetone containing 2% uranyl acetate, followed by warming to −50°C and embedding in Lowicryl HM20 gave consistent and clear membrane detail even when imaged without section contrasting. Preliminary data indicates that the high intrinsic contrast of samples prepared in this way will be valuable for tomographic studies. Immunolabelling sensitivity of sections of samples prepared by this rapid substitution technique was poor; however, reducing the uranyl acetate concentration in the substitution medium to 0.2% resulted in improved labelling. Samples substituted in this lower concentration of uranyl acetate also gave good membrane detail when imaged after section contrasting.

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참고문헌 (22)

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Rapid freeze-substitution preserves membranes in high-pressure frozen tissue culture cells

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