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首页> 外文期刊>Brain research. Brain research protocols >High-pressure freezing followed by cryosubstitution as a tool for preserving high-quality ultrastructure and immunoreactivity in the Xenopus laevis pituitary gland.
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High-pressure freezing followed by cryosubstitution as a tool for preserving high-quality ultrastructure and immunoreactivity in the Xenopus laevis pituitary gland.

机译：高压冷冻然后冷冻替代是在非洲爪蟾垂体中用于维持高质量超微结构和免疫反应性的工具。

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摘要

Subcellular localisation of proteins and peptides yields fundamental information about cell functioning. Immunoelectron microscopy is a powerful tool to achieve this goal, but combining good tissue preservation with strong immunoreactivity is a great challenge in electron microscopy. We have applied a novel approach, using high-pressure freezing (HPF) followed by cryosubstitution, to prepare the pituitary gland of the amphibian Xenopus laevis for immunogold-electron microscopy. In this way, we investigated the subcellular distribution of brain-derived neurotrophic factor and the amphibian neurohormone mesotocin in the pituitary neural lobe, and the peptide hormone alpha-melanophore-stimulating hormone and its protein precursor proopiomelanocortin in melanotrope cells of the pituitary intermediate lobe. In contrast to conventional chemical fixation (followed by cryosubstitution), HPF not only revealed strong immunoreactivity of the secretory products, but also provided excellent ultrastructural preservation of cell organelles, including secretory granule subtypes. We conclude that HPF followed by cryosubstitution provides a preparation technique of choice when both optimal tissue ultrastructure and strong immunoreactivity are required.
机译：蛋白质和多肽的亚细胞定位可产生有关细胞功能的基本信息。免疫电子显微镜是实现这一目标的有力工具，但是将良好的组织保存与强大的免疫反应性相结合是电子显微镜面临的巨大挑战。我们已经应用了一种新颖的方法，先用高压冷冻（HPF）再冷冻替换，以制备用于免疫金电子显微镜检查的两栖非洲爪蟾的垂体。通过这种方式，我们研究了脑源性神经营养因子和两栖神经激素mesotocin在垂体神经叶中的亚细胞分布，以及在垂体中叶黑素体细胞中的肽激素α-黑素细胞刺激激素及其蛋白前体proopiomelanocortin。与常规化学固定法不同（后继冷冻替代法），HPF不仅显示出分泌产物的强免疫反应性，而且还为细胞器包括分泌颗粒亚型提供了出色的超微结构保存。我们得出的结论是，当同时需要最佳的组织超微结构和强免疫反应性时，HPF继而进行低温替代可以提供一种选择的制备技术。

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