High‐pressure freezing for immunocytochemistry

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Abstract
Ultrastructural immunocytochemistry requires that minimal damage to antigens is imposed by the processing methods. Immersion fixation in cross‐linking fixatives with their potential to damage antigens is not an ideal approach and rapid freezing as an alternative sample‐stabilization step has a number of advantages. Rapid freezing at ambient pressure restricts the thickness of well‐frozen material obtainable to ≈ 15 μm or less. In contrast, high‐pressure freezing has been demonstrated to provide ice‐crystal‐artefact‐free freezing of samples up to 200 μm in thickness. There have been few reports of high‐pressure freezing for immunocytochemical studies and there is no consensus on the choice of post‐freezing sample preparation. A range of freeze‐substitution time and temperature protocols were compared with improved tissue architecture as the primary goal, but also to compare ease of resin‐embedding, polymerization and immunocytochemical labelling. Freeze‐substitution in acetone containing 2% osmium tetroxide followed by epoxy‐resin embedding at room temperature gave optimum morphology. Freeze‐substitution in methanol was completed within 18 h and in tetrahydrofuran within 48 h but the cellular morphology of the Lowicryl‐embedded samples was not as good as when samples were substituted in pure acetone. Acetone freeze‐substitution was slow, taking at least 6 days to complete, and gave blocks which were difficult to embed in Lowicryl HM20. Careful handling of frozen samples avoiding rapid temperature changes reduced apparent ice‐crystal damage in sections of embedded material. Thus a slow warm‐up to freeze‐substitution temperature and a long substitution time in acetone gave the best results in terms of freezing quality and cellular morphology. No clear differences emerged between the different freeze‐substitution media from immunocytochemical labelling experiments.

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