Authors: Feng Zhou, Qiaowei Tang, Xin Yan, Chao Ma, Yu Zhang, Jichao Zhang, Qian Li, Lihua Wang, Jun Hu, Xiaoqing Cai, Jiang Li, Ying Zhu, Chunhai Fan
Published: 2025-05-28
Source: Full article
AbstractAchieving detailed neuronal structural information in large‐volume brain tissue has been a longstanding challenge in human brain imaging. A key obstacle arises from the trade‐off between staining efficiency and tissue autolysis. Traditional Golgi staining, typically conducted at room temperature or 37 °C to optimize staining efficiency, leads to rapid autolysis of brain tissue, resulting in the loss of fine structural details. Here, a near‐freezing temperature (NFT) staining strategy in post‐mortem frozen (PMF) human brain samples are presented, using a mercury chloride‐based method under ice‐water bath conditions. In contrast to the 37 °C Golgi staining, this NFT‐based method significantly reduces tissue autolysis, preserving fine neuronal structures. Notably, neuronal counts in the same field of view increased by 5.5‐fold, and dendritic spine density increases by 22‐fold. Using this approach, uniform staining of millimeter‐thick is achieved, centimeter‐scale human brain slices and integrated it with synchrotron‐based X‐ray microscopy to perform micrometer resolution 3D reconstructions of the cerebellum and frontal lobe. This novel technique offers a powerful tool for the fine‐structural imaging of large‐volume brain tissue, providing new insights into the intricate organization of neural networks.