High‐pressure freezing for immunocytochemistry

Abstract

SummaryUltrastructural 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 Ϸ 15 mm or less. In contrast, high-pressure freezing has been demonstrated to provide ice-crystalartefact-free freezing of samples up to 200 mm 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 freezesubstitution 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.

Ultrastructural and nuclear antigen preservation after high-pressure freezing/freeze-substitution and low-temperature LR White embedding of HeLa cells

“…1a). Also the lowered contrast is in agreement with several works mentioning lower contrast after using acrylic resins (Nicolas and Bassot 1993;Roch et al 1997;von Schack et al 1991von Schack et al , 1993Monaghan et al 1998). This feature, however, can be sometimes an advantage since gold particles become better visible over the cellular structures.…”

“…For immunocytochemical studies, Lowicryl resins have been the most widely used acrylic resins because they allow for embedding of the samples at very low temperatures, and they preserve well the ultrastructural details of biological samples (for a few examples, see Roth et al 1989;Bittermann et al 1992;Schwarz et al 1993;Monaghan et al 1998;McDonald 1999;Bohrmann and Kellenberger 2001;Giddings 2003;Reipert et al 2004a;Sawaguchi et al 2004;Mühlfeld and Richter 2006). Despite their disadvantageous toxicity and strong allergenic properties, they are usually the resins of Wrst choice.…”

The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications

“…HPF has been previously combined with immunolabeling (Donohoe et al, 2007;McDonald, 1999;Mobius et al, 2002;Monaghan et al, 1998;Nicolas et al, 1997) and in particular has been applied to brain tissue focusing on myelin glycolipids (Kirschning et al, 1998), cytoskeletal tethers in rat hippocampal PSDs (Rostaing et al, 2006;Siksou et al, 2007), kainate receptors in the monkey striatum (Kieval et al, 2001), GABA receptors in the monkey subthalamic nucleus (Galvan et al, 2004) and neurohormonal/secretory activity in neuroendocrine melanotrope cells (Calle et al, 2005;Wang et al, 2004). In several of these studies (e.g.…”

Architecture of Thylakoid Membrane Networks

“…Freeze-substituted samples, when compared to chemically fixed ones, lack some of the artifacts attributed to CF and are considerably more wellpreserved -their membranes appear smoother and less undulated, organelles, especially in plant tissues, do not tend to detach from their surroundings (Studer et al, 1992), distortions are absent (Szczesny et al, 1996;Murk et al, 2003) and lipid extraction is limited (Pfeiffer et al, 2000), as opposed to CF (Maneta-Peyret et al, 1999). FS is also an optimal method for preserving the antigenicity of a sample without a large sacrifice of structural fidelity (Steinbrecht and Müller, 1987;Monaghan et al, 1998). However, to some extent, samples processed by HPF-FS may suffer from aggregation artifacts during water removal (Dubochet and Blanc, 2001).…”