An improved procedure for low-temperature embedding of high-pressure frozen and freeze-substituted plant tissues resulting in excellent structural preservation and contrast

Here we describe refinements in the processing of high-pressure frozen samples of delicate plant tissues for immuno-electron microscopy. These involve: shortened freeze-substitution schedules, lower temperatures during processing and polymerisation, the avoidance of temperature fluctuations and the optimisation of heat transfer from the specimens using small disposable aluminium containers. The application of these modifications leads to very good structural preservation and selective membrane contrast. As a result, the versatility of the method is increased since not only immuno-electron microscopical studies can be performed but often the quality is also quite suitable for structural investigations.