CLARITY increases sensitivity and specificity of fluorescence immunostaining in long-term archived human brain tissue.
CLARITY increases sensitivity and specificity of fluorescence immunostaining in long-term archived human brain tissue.
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DOI：
10.1186/s12915-023-01582-6
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发表时间：
2023-05-24
期刊：
影响因子：
5.4
通讯作者：
Schön M
中科院分区：
文献类型：
--
作者：
Woelfle S;Deshpande D;Feldengut S;Braak H;Del Tredici K;Roselli F;Deisseroth K;Michaelis J;Boeckers TM;Schön M
死后的人类脑组织是研究细胞类型、连通性以及亚细胞结构直至中枢神经系统的分子设置的重要资源，特别是涉及过多的脑部疾病。一种关键的方法是用荧光染料进行免疫染色，它可以同时在多个结构的三维上进行高分辨率成像。虽然收集了大量用福尔马林固定的大脑标本，但研究往往受到限制，因为出现了一些情况，使使用人脑组织进行高分辨率荧光显微镜变得复杂。在这项研究中，我们开发了一种清除方法，用于基于免疫荧光的分析灌注和浸泡固定的死后人脑组织，称为人类透明脂质交换丙烯酰胺杂交刚性成像/免疫染色/原位杂交兼容组织水凝胶（hCLARITY）。hCLARITY通过减少脱靶标记来优化特异性，并在人脑切片中产生非常敏感的染色，允许超分辨率显微镜对突触前和突触后区室进行前所未有的成像。此外，hCLARITY保留了阿尔茨海默病的特征，重要的是经典的3,3 ' -二氨基联苯胺（DAB）或尼氏染色与该方案兼容。hCLARITY是非常通用的，使用了超过30种性能良好的抗体，并允许对同一组织切片进行分离和随后的重新染色，这对于多标记方法非常重要，例如在超分辨率显微镜中。总之，hCLARITY能够以高灵敏度和低至亚衍射分辨率研究人脑。因此，它对局部形态学变化的研究具有巨大的潜力，例如，在神经退行性疾病中。在线版本包含补充材料，可在10.1186/s12915-023-01582-6获得。
Post mortem human brain tissue is an essential resource to study cell types, connectivity as well as subcellular structures down to the molecular setup of the central nervous system especially with respect to the plethora of brain diseases. A key method is immunostaining with fluorescent dyes, which allows high-resolution imaging in three dimensions of multiple structures simultaneously. Although there are large collections of formalin-fixed brains, research is often limited because several conditions arise that complicate the use of human brain tissue for high-resolution fluorescence microscopy. In this study, we developed a clearing approach for immunofluorescence-based analysis of perfusion- and immersion-fixed post mortem human brain tissue, termed human Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging / Immunostaining / In situ hybridization-compatible Tissue-hYdrogel (hCLARITY). hCLARITY is optimized for specificity by reducing off-target labeling and yields very sensitive stainings in human brain sections allowing for super-resolution microscopy with unprecedented imaging of pre- and postsynaptic compartments. Moreover, hallmarks of Alzheimer’s disease were preserved with hCLARITY, and importantly classical 3,3’-diaminobenzidine (DAB) or Nissl stainings are compatible with this protocol. hCLARITY is very versatile as demonstrated by the use of more than 30 well performing antibodies and allows for de- and subsequent re-staining of the same tissue section, which is important for multi-labeling approaches, e.g., in super-resolution microscopy. Taken together, hCLARITY enables research of the human brain with high sensitivity and down to sub-diffraction resolution. It therefore has enormous potential for the investigation of local morphological changes, e.g., in neurodegenerative diseases. The online version contains supplementary material available at 10.1186/s12915-023-01582-6.
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影响因子：
16.2
作者：
Dani, Adish;Huang, Bo;Bergan, Joseph;Dulac, Catherine;Zhuang, Xiaowei
通讯作者：
Zhuang, Xiaowei
影响因子：
5.3
作者：
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通讯作者：
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影响因子：
14
作者：
de Wilde, Martijn C.;Overk, Cassia R.;Sijben, John W.;Masliah, Eliezer
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Masliah, Eliezer
影响因子：
14.5
作者：
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通讯作者：
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影响因子：
2.3
作者：
Grillo, Federica;Pigozzi, Simona;Mastracci, Luca
通讯作者：
Mastracci, Luca