Cryofixation by high‐pressure freezing (HPF) followed by freeze substitution (FS) is a preferred method to prepare biological specimens for ultrastructural studies. It has been shown to achieve uniform vitrification and ultrastructure preservation of complex structures in different cell types. One limitation of HPF is the small sample volume of <200 µm thickness and about 2000 µm across. A wool follicle is a rare intact organ in a single sample about 200 µm thick. Within each follicle, specialized cells derived from multiple cell lineages assemble, mature and cornify to make a wool fibre, which contains 95% keratin and associated proteins. In addition to their complex structure, large density changes occur during wool fibre development. Limited water movement and accessibility of fixatives are some issues that negatively affect the preservation of the follicle ultrastructure via conventional chemical processing. Here, we show that HPF‐FS of wool follicles can yield high‐quality tissue preservation for ultrastructural studies using transmission electron microscopy.

中文翻译：

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高压冷冻，然后冷冻替代复杂且密度可变的微型器官：毛囊

高压冷冻 (HPF) 冷冻固定和冷冻置换 (FS) 是制备用于超微结构研究的生物标本的首选方法。它已被证明可以实现不同细胞类型中复杂结构的均匀玻璃化和超微结构保存。HPF 的一个限制是样品体积小，厚度小于 200 µm，宽度约为 2000 µm。毛囊是单个样本中罕见的完整器官，厚度约为 200 µm。在每个毛囊内，来自多个细胞谱系的特化细胞组装、成熟和角质化，形成羊毛纤维，其中包含 95% 的角蛋白和相关蛋白质。除了其复杂的结构外，羊毛纤维发育过程中还会发生较大的密度变化。有限的水运动和固定剂的可及性是一些问题，通过传统的化学处理对毛囊超微结构的保存产生负面影响。在这里，我们表明羊毛毛囊的 HPF-FS 可以为使用透射电子显微镜的超微结构研究提供高质量的组织保存。