Department of Health, Human Performance, and Recreation, Baylor University, 254-710-2911, B.207 Baylor Science Building, One Bear Place #97388, 76798-7388, Waco, TX 76706, USA.
Department of Biology, Baylor University, Waco, TX 76706, USA.
Ultramicroscopy. 2022 Nov;241:113600. doi: 10.1016/j.ultramic.2022.113600. Epub 2022 Aug 11.
Muscle samples are commonly chemically fixed or frozen immediately upon collection for biochemical and morphological analysis. Certain fixatives such as glutaraldehyde and osmium tetroxide are widely used for transmission electron microscopy (TEM) and lead to adequate preservation of muscle ultrastructure, but do not preserve the molecular features of samples. Methacarn is suggested to be a preferable chemical fixative for light microscopy because it maintains immunohistological features of samples. However, the efficacy of methacarn to preserve ultrastructural features as a primary chemical fixative for TEM is currently unclear. Additionally, cryo-preservation of samples for TEM analysis involves freezing processes such as plunge freezing, slam freezing, or high pressure freezing. High pressure freezing is the considered the gold standard but requires costly equipment and may not be a viable option for many labs collecting tissue samples from remote locations. Dimethyl sulfoxide (DMSO) is a commonly used cryoprotectant that may allow for better structural preservation of samples by impairing ice damage that occurs during plunge/snap freezing. We aimed to assess the effectiveness of methacarn as a primary chemical fixative and determine the effect of pre-coating samples with DMSO before plunge/snap freezing tissues to be prepared for TEM. The micrographs of the methcarn-fixed samples indicate a loss of Z-disk integrity, intermyofibrillar space, mitochondria structure, and lipids. Ultimately, methacarn is not a viable primary fixative for tissue sample preparation for TEM. Similarly, liquid nitrogen freezing of samples wrapped in aluminum foil produced non-uniform Z-disk alignments that appeared smeared with swollen mitochondria. DMSO coating before freezing appears to lessen the alterations to contractile and mitochondrial morphological structures. DMSO appears to be useful for preserving the ultrastructure of sarcomeres if samples are covered before freezing.
肌肉样本通常在采集后立即进行化学固定或冷冻，以便进行生化和形态分析。某些固定剂，如戊二醛和四氧化锇，广泛用于透射电子显微镜（TEM），可充分保存肌肉超微结构，但不能保存样本的分子特征。甲咔烷被建议作为光镜的首选化学固定剂，因为它能保持样本的免疫组织化学特征。然而，作为 TEM 的主要化学固定剂，甲咔烷保持超微结构特征的效果目前尚不清楚。此外，为了进行 TEM 分析而对样本进行冷冻保存涉及冷冻过程，如 plunge freezing、slam freezing 或 high pressure freezing。高压冷冻被认为是黄金标准，但需要昂贵的设备，并且对于许多从偏远地区采集组织样本的实验室来说可能不是可行的选择。二甲亚砜（DMSO）是一种常用的冷冻保护剂，通过削弱 plunge/snap 冷冻过程中发生的冰晶损伤，可能允许更好地保存样本的结构。我们旨在评估甲咔烷作为主要化学固定剂的效果，并确定在 plunge/snap 冷冻组织之前用 DMSO 对样本进行预包被以准备 TEM 的效果。甲咔烷固定样本的显微照片显示 Z 盘完整性、肌原纤维间空间、线粒体结构和脂质丧失。最终，甲咔烷不是用于 TEM 组织样本制备的可行主要固定剂。同样，用铝箔包裹的样本的液氮冷冻产生了不均匀的 Z 盘排列，看起来线粒体肿胀且排列模糊。冷冻前的 DMSO 涂层似乎减轻了收缩和线粒体形态结构的改变。如果在冷冻前覆盖样本，DMSO 似乎对保存肌节的超微结构有用。
