Rosen Grace A, Kirsch Daniel, Nicks Raymond, Kelley Hunter, Mathias Rebecca, Cormier Kerry A, Kubilus Caroline A, Dec Bryan, Stein Thor D, Alvarez Victor E, Alosco Michael L, McKee Ann C, Huber Bertrand R
VA Boston Healthcare System, US Department of Veterans Affairs, Boston, MA, United States.
Department of Neurology, Boston University Chobanian and Avedisian School of Medicine, Boston, MA, United States.
Front Neurosci. 2024 Oct 9;18:1474617. doi: 10.3389/fnins.2024.1474617. eCollection 2024.
Postmortem human brain tissue is a critical resource for studying neurodegenerative disease, providing critical insights into cellular morphology, pathology, and network connectivity. To improve standard microscopy and enable high-resolution, three-dimensional (3D) images of tissues at the subcellular level, tissue-clearing methods have been developed. These 3D images allow for the analysis of large regions of interest and can be used to study structural and spatial changes that occur during neurodegeneration. Additionally, 3D imaging facilitates the visualization of whole-cell morphology, especially in cells with long processes that would otherwise be truncated in single-plane images. Human brain tissue is especially challenging for tissue clearing due to the abundance of lipids in myelin and the need for optimal fixation and low postmortem intervals. Formaldehyde-based fixatives, commonly used in preserving tissue, hinder antibody binding by crosslinking important antibody epitopes, and fluorescent microscopy requires the incorporation of fluorescent labels through passive diffusion or electrophoresis. Recent studies have focused on optimally fixed human brain tissue with short postmortem intervals, limiting the general applicability of these methods. To address these challenges, we developed SHARD (SHIELD, antigen retrieval, and delipidation), a simple and widely applicable method for clearing and labeling human brain tissue, which can be applied to long-term banked human brain tissue preserved in formaldehyde. SHARD is a novel addition to the SHIELD tissue clarification method, combining antigen retrieval, tissue clearing, and staining of 200-μm sections from long-term banked human brain tissue. The SHARD method is effective for postmortem intervals (PMIs) ranging from 10 to 72 h in multiple neurodegenerative diseases and control samples. In this study, we demonstrate that the SHARD method significantly enhances the immunostaining of glial fibrillary acidic protein (GFAP), an astrocytic cytoskeletal marker. Overall, the combination of antigen retrieval and tissue delipidation holds great potential for achieving detailed 3D immunostaining in long-term formaldehyde-fixed postmortem human brain tissue, opening new avenues for research and discovery.
尸检人脑组织是研究神经退行性疾病的关键资源，能为细胞形态学、病理学和网络连通性提供重要见解。为改进标准显微镜检查并实现亚细胞水平组织的高分辨率三维（3D）图像，已开发出组织透明化方法。这些3D图像可用于分析大的感兴趣区域，并可用于研究神经退行性变过程中发生的结构和空间变化。此外，3D成像有助于可视化全细胞形态，特别是对于那些具有长突起的细胞，否则在单平面图像中这些突起会被截断。由于髓磷脂中脂质丰富，且需要最佳固定和较短的死后间隔时间，人脑组织的组织透明化尤其具有挑战性。常用于保存组织的基于甲醛的固定剂会通过交联重要的抗体表位来阻碍抗体结合，而荧光显微镜检查需要通过被动扩散或电泳掺入荧光标记。最近的研究集中在死后间隔时间短且固定良好的人脑组织上，限制了这些方法的普遍适用性。为应对这些挑战，我们开发了SHARD（屏蔽、抗原修复和脱脂），这是一种简单且广泛适用的用于人脑组织透明化和标记的方法，可应用于保存在甲醛中的长期保存的人脑组织。SHARD是对SHIELD组织透明化方法的新补充，结合了抗原修复、组织透明化以及对长期保存的人脑组织200μm切片的染色。SHARD方法对于多种神经退行性疾病和对照样本中10至72小时的死后间隔时间（PMI）有效。在本研究中，我们证明SHARD方法显著增强了胶质纤维酸性蛋白（GFAP）的免疫染色，GFAP是一种星形细胞细胞骨架标记物。总体而言，抗原修复和组织脱脂的结合在长期甲醛固定的尸检人脑组织中实现详细的3D免疫染色方面具有巨大潜力，为研究和发现开辟了新途径。
