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

• Published in: Journal of microscopy (Oxford) Vol. 226; no. 2; pp. 182 - 189
• Main Authors: HAWES, P, NETHERTON, C.L, MUELLER, M, WILEMAN, T, MONAGHAN, P
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.05.2007
• Subjects: African swine fever virus; Animals; Cells, Cultured; Cercopithecus aethiops; Cricetinae; Freeze Substitution - methods; freeze-substitution; Freezing; high-pressure freezing; Hydrostatic Pressure; Immunohistochemistry; immunolabelling; Lowicryl; Microscopy, Immunoelectron; Organometallic Compounds; tissue culture; Vero Cells
• ISSN: 0022-2720; 1365-2818
• DOI: 10.1111/j.1365-2818.2007.01767.x

We 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.