Gottfried Schatz Research Center for Cell Signaling, Metabolism and Aging, Division of Cell Biology, Histology and Embryology, Medical University of Graz, Neue Stiftingtalstraße 6/II, 8010, Graz, Austria.
Otto Loewi Research Center for Vascular Biology, Immunology and Inflammation, Division of Physiology, LBI for Lung Vascular Research, Medical University of Graz, Stiftingtalstrasse 24, 8010, Graz, Austria.
Histochem Cell Biol. 2021 May;155(5):593-603. doi: 10.1007/s00418-020-01952-z. Epub 2021 Jan 6.
Preservation of ultrastructural features in biological samples for electron microscopy (EM) is a challenging task that is routinely accomplished through chemical fixation or high-pressure freezing coupled to automated freeze substitution (AFS) using specialized devices. However, samples from clinical (e.g. "biobanking" of bulk biopsies) and preclinical (e.g. whole mouse tissues) specimens are often not specifically prepared for ultrastructural analyses but simply immersed in liquid nitrogen before long-term cryo-storage. We demonstrate that ultrastructural features of such samples are insufficiently conserved using AFS and developed a simple, rapid, and effective method for thawing that does not require specific instrumentation. This procedure consists of dry ice-cooled pre-trimming of frozen tissue and aldehyde fixation for 3 h at 37 °C followed by standard embedding steps. Herein investigated tissues comprised human term placentae, clinical lung samples, as well as mouse tissues of different composition (brown adipose tissue, white adipose tissue, cardiac muscle, skeletal muscle, liver). For all these tissues, we compared electron micrographs prepared from cryo-stored material with our method to images derived from directly prepared fresh tissues with standard chemical fixation. Our protocol yielded highly conserved ultrastructural features and tissue-specific details, largely matching the quality of fresh tissue samples. Furthermore, morphometric analysis of lipid droplets and mitochondria in livers of fasted mice demonstrated that statistically valid quantifications can be derived from samples prepared with our method. Overall, we provide a simple and effective protocol for accurate ultrastructural and morphometric analyses of cryo-stored bulk tissue samples.
为了进行电子显微镜（EM）的超微结构研究，生物样本的保存是一项极具挑战性的任务，通常通过化学固定或高压冷冻结合自动化冷冻置换（AFS），并使用专门的设备来实现。然而，来自临床（例如，大容量活检的“生物库”）和临床前（例如，整个小鼠组织）标本的样本通常没有专门为超微结构分析准备，而是简单地浸入液氮中进行长期低温储存。我们证明，使用 AFS 对这些样本的超微结构特征的保存是不充分的，并开发了一种简单、快速和有效的解冻方法，该方法不需要特定的仪器。该程序包括用干冰冷却冷冻组织的预修剪，然后在 37°C 下用醛固定 3 小时，接着进行标准包埋步骤。在此研究中，所涉及的组织包括人足月胎盘、临床肺样本以及不同组成的小鼠组织（棕色脂肪组织、白色脂肪组织、心肌、骨骼肌、肝脏）。对于所有这些组织，我们将从冷冻储存材料中制备的电子显微镜图像与我们的方法与直接从新鲜组织中制备的图像进行了比较，这些新鲜组织使用标准化学固定。我们的方案得到了高度保存的超微结构特征和组织特异性细节，与新鲜组织样本的质量基本匹配。此外，对禁食小鼠肝脏中脂质滴和线粒体的形态计量分析表明，从我们的方法制备的样本中可以得出具有统计学意义的定量结果。总体而言，我们为准确的超微结构和形态计量分析冷冻储存的大块组织样本提供了一种简单有效的方案。
