Eye Research Institute, Oakland University.
Eye Research Institute, Oakland University;
J Vis Exp. 2021 Mar 6(169). doi: 10.3791/62178.
The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining and imaging of thick brain samples. However, this advanced technology has not yet been used for whole-mount retinas. Although the retina is partially transparent, its thickness of approximately 200 µm (in mice) still limits the penetration of antibodies into the deep tissue as well as reducing light penetration for high-resolution imaging. Here, we adapted the CLARITY method for whole-mount mouse retinas by polymerizing them with an acrylamide monomer to form a nanoporous hydrogel and then clearing them in sodium dodecyl sulfate to minimize protein loss and avoid tissue damage. CLARITY-processed retinas were immunostained with antibodies for retinal neurons, glial cells, and synaptic proteins, mounted in a refractive index matching solution, and imaged. Our data demonstrate that CLARITY can improve the quality of standard immunohistochemical staining and imaging for retinal neurons and glial cells in whole-mount preparation. For instance, 3D resolution of fine axon-like and dendritic structures of dopaminergic amacrine cells were much improved by CLARITY. Compared to non-processed whole-mount retinas, CLARITY can reveal immunostaining for synaptic proteins such as postsynaptic density protein 95. Our results show that CLARITY renders the retina more optically transparent after the removal of lipids and preserves fine structures of retinal neurons and their proteins, which can be routinely used for obtaining high-resolution imaging of retinal neurons and their subcellular structures in whole-mount preparation.
组织水凝胶去脂方法（CLARITY）最初由 Deisseroth 实验室开发，经过改良后已被广泛用于厚脑样本的免疫染色和成像。然而，这项先进技术尚未用于全视网膜。尽管视网膜部分透明，但它约 200 µm 的厚度（在小鼠中）仍然限制了抗体进入深层组织的穿透能力，并降低了用于高分辨率成像的光穿透率。在这里，我们通过将丙烯酰胺单体聚合到全视网膜小鼠中以形成纳米多孔水凝胶，然后用十二烷基硫酸钠对其进行处理来清除水凝胶，从而最小化蛋白质损失并避免组织损伤，从而使 CLARITY 方法适用于全视网膜。用针对视网膜神经元、神经胶质细胞和突触蛋白的抗体对 CLARITY 处理的视网膜进行免疫染色，将其安装在折射率匹配溶液中，并进行成像。我们的数据表明，CLARITY 可以改善全视网膜制剂中视网膜神经元和神经胶质细胞的标准免疫组织化学染色和成像质量。例如，多巴胺能无长突细胞的精细轴突样和树突状结构的 3D 分辨率通过 CLARITY 得到了很大的改善。与未经处理的全视网膜相比，CLARITY 可以显示突触蛋白（如突触后密度蛋白 95）的免疫染色。我们的结果表明，CLARITY 在去除脂质后使视网膜更加光学透明，并保留了视网膜神经元及其蛋白质的精细结构，这可以常规用于获得全视网膜制剂中视网膜神经元及其亚细胞结构的高分辨率成像。
