Freeze-fracturing and deep-etching with the volatile cryoprotectant ethanol reveals true membrane surfaces of kidney structures.

With the conventional freeze-fracture technique applied to biological specimens, cell membranes split along an interior plane and two membrane faces are produced. True membrane surfaces remain hidden and can only be uncovered by deep-etching. To date, deep-etching could not be satisfactorily performed in the presence of cryoprotective agents since conventional cryoprotectants do not sublime due to their low vapour pressure. This lack of suitable volatile cryoprotectants has limited deep-etching so far to very small objects which can be cryofixed without cryoprotectants. As a consequence, our freeze-fracture knowledge of cell surfaces is still poor. The present study shows that ethanol is a suitable volatile cryoprotectant for the freeze-fracture technique, and provides a novel approach to the routine deep-etching of freeze-fracture specimens without the need for special equipment. With ethanol deep-etching, true outer cell-surfaces are demonstrated within the kidneys of rat and Psammomys.