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Freeze-substitution and transmission electron microscopy
Trichoplax adhaerens were prepared by freeze-substitution, as previously described (Smith et al., 2014).
Rapid freeze-substitution preserves membranes in high-pressure frozen tissue culture cells.
Cryofixation and ensuing freeze-substitution at ambient (Notzli & Clark, 1994) or high pressure (Hunziker & Schenck, 1984; Keene & McDonald, 1993; Engfeldt et al., 1994) is possible.
The cryofixation, entailing freezing at ambient pressure followed by freeze-substitution, allowed a rapid preservation of the initial state within seconds, bit the ensuing fixation with the chemical substitution took several days.
A quick-freeze and freeze-substitution method was mainly used for examination of cytoskeletons, and chemical fixation was used for examination of other structures.
In the specimens prepared by the quick-freeze and freeze-substitution method, bundles of fibers could be identified within microvilli in immature oocytes (Fig.
We could not examine microfilaments in the fixing processes by transmission electron microscopy, because the fixing processes were not preserved by the quick-freeze and freeze-substitution fixation that effectively preserves microfilaments.
Frozen tissues were transferred to liquid [N.sub.2] and held there briefly before being transferred into a Leica EM AFS automatic freeze-substitution system.
When the technique of freeze-substitution was used to fix tissue from Cassiopeia xamachana, Aiptasia pallida, and Phyllactis flosculifera and prepare it for embedding, thecal vesicles were revealed within the in situ symbionts of all three species.
In the following paper, we further amend the ultrastructural description of the genus Symbiodinium on the basis of the use of freeze-substitution, an alternative fixation technique that provides better preservation of fine structure.