Zeng Fu, Huang Lian, Han Ding, Liu Renjie, Zhang Kongjia, Su Yuwen, Su Tong, Lin Yarong, Xiu Jianbo
State Key Laboratory of Common Mechanism Research for Major Diseases, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing 100005, China.
Neuroscience Center, Chinese Academy of Medical Sciences, Beijing 100005, China.
Int J Mol Sci. 2025 Apr 10;26(8):3569. doi: 10.3390/ijms26083569.
Recent advances in tissue clearing chemistry have revolutionized three-dimensional imaging by enabling whole-organ antibody labeling, even in thick human tissue samples. However, these techniques face limitations, including reduced clearance efficiency in thick tissue blocks, prolonged processing times, and other challenges specific to formalin-fixed human brain tissue, such as autofluorescence due to the presence of lipofuscin, neuromelanin pigments, and residual blood. To address these challenges, we have developed an improved SHANEL procedure, iSHANEL, which is compatible with brain tissue from multiple species, including nonrodents. iSHANEL significantly enhances the tissue transparency. It effectively removes lipids, particularly sphingolipids, from brain tissue across different species. For brain vasculature imaging, it offers the better visualization of vascular structures at high magnification, reducing light scattering and background noise. Moreover, iSHANEL can be effectively applied to large tissue from pigs and cynomolgus monkeys, and it preserves protein immunogenicity well, as evidenced by immunostaining with the microglia-specific marker IBA-1. We also investigated methods to reduce blood vessel autofluorescence. Overall, iSHANEL provides an improved procedure for the high-resolution 3D imaging of brain tissue across multiple species.
组织透明化化学领域的最新进展彻底改变了三维成像技术，通过实现全器官抗体标记，即使在厚的人体组织样本中也是如此。然而，这些技术面临着一些限制，包括厚组织块中清除效率降低、处理时间延长以及福尔马林固定的人脑组织特有的其他挑战，如由于脂褐素、神经黑素色素和残留血液的存在而产生的自发荧光。为了应对这些挑战，我们开发了一种改进的SHANEL程序，即iSHANEL，它与包括非啮齿动物在内的多种物种的脑组织兼容。iSHANEL显著提高了组织透明度。它能有效去除不同物种脑组织中的脂质，特别是鞘脂。对于脑血管成像，它在高倍放大下能更好地显示血管结构，减少光散射和背景噪声。此外，iSHANEL可以有效地应用于猪和食蟹猴的大型组织，并且能很好地保留蛋白质免疫原性，小胶质细胞特异性标志物IBA-1的免疫染色证明了这一点。我们还研究了减少血管自发荧光的方法。总体而言，iSHANEL为跨多种物种的脑组织高分辨率三维成像提供了一种改进的程序。
