Optical clearing techniques provide unprecedented opportunities to study large tissue samples at histological resolution, eliminating the need for physical sectioning while preserving the three-dimensional structure of intact biological systems. There is significant potential for applying optical clearing to reproductive tissues. In testicular biology, for example, the study of spermatogenesis and the use of spermatogonial stem cells offer high-impact applications in fertility medicine and reproductive biotechnology. The objective of our study is to apply optical clearing, immunofluorescence, and confocal microscopy to testicular tissue in order to reconstruct its three-dimensional microstructure in intact samples. We used Triton-X/DMSO clearing in combination with refractive index matching to achieve optical transparency of fixed mouse testes. An antibody against smooth muscle actin was used to label peritubular myoid cells of seminiferous tubules while an antibody against ubiquitin C-terminal hydrolase was used to label Sertoli cells and spermatogonia in the seminiferous epithelium. Specimens were then imaged using confocal fluorescence microscopy. We were able to successfully clear testicular tissue and utilize immunofluorescent probes. Additionally, we successfully visualized the histological compartments of testicular tissue in three-dimensional reconstructions. Optical clearing combined with immunofluorescence and confocal imaging offers a powerful new method to analyze the cytoarchitecture of testicular tissue at histological resolution while maintaining the macro-scale perspective of the intact system. Considering the importance of the murine model, our developed method represents a significant contribution to the field of male reproductive biology, enabling the study of testicular function.

中文翻译：

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清除，免疫荧光和共聚焦显微镜，用于小鼠睾丸的三维成像和睾丸生物学研究。

光学清除技术为以组织学分辨率研究大型组织样本提供了前所未有的机会，从而无需进行物理切片，同时保留了完整生物系统的三维结构。将光学清除应用于生殖组织具有巨大的潜力。例如，在睾丸生物学中，精子发生的研究和精原干细胞的使用在生育医学和生殖生物技术中具有重要影响。我们研究的目的是将光学清除，免疫荧光和共聚焦显微镜应用于睾丸组织，以便在完整样品中重建其三维显微结构。我们将Triton-X / DMSO清除与折射率匹配结合使用，以实现固定小鼠睾丸的光学透明性。抗平滑肌肌动蛋白的抗体被用来标记生精小管的肾小管周肌细胞，而抗泛素C末端水解酶的抗体被用来标记生精上皮中的支持细胞和精原细胞。然后使用共聚焦荧光显微镜对标本成像。我们能够成功清除睾丸组织并利用免疫荧光探针。此外，我们成功地在三维重建中可视化了睾丸组织的组织学区室。光学清除结合免疫荧光和共聚焦成像提供了一种强大的新方法，可以在组织学分辨率下分析睾丸组织的细胞结构，同时保持完整系统的宏观视角。考虑到鼠模型的重要性，