Journal Article

Near‐Freezing‐Temperature Golgi Neuronal Staining for X‐ray Imaging of Human Brain

Zhang, Jichao, Zhu, Ying, Hu, Jun, Tang, Qiaowei, Zhang, Yu, Cai, Xiaoqing, Li, Qian, Zhou, Feng, Ma, Chao, Yan, Xin, Li, Jiang, Wang, Lihua, Fan, Chunhai

2025

Overview

Achieving 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. A near‐freezing temperature staining strategy, combined with a mercury chloride‐based method, effectively preserves delicate neuronal structures in post‐mortem frozen human brain tissues by minimizing autolysis. This approach enables uniform labeling across centimeter‐scale slices and, when integrated with synchrotron‐based X‐ray microscopy, achieves micrometer‐resolution 3D reconstructions, paving the way for large‐scale human brain mapping.

• Publisher: John Wiley & Sons, Inc,John Wiley and Sons Inc,Wiley
• Subject: Brain / Brain - diagnostic imaging / Freezing / Humans / Imaging, Three-Dimensional - methods / mercury chloride (HgCl2) / Metabolism / Microscopy / Morphology / near‐freezing‐temperature (NFT) Golgi / Neuroimaging - methods / neuron / Neurons / post‐mortem frozen human brain (PMF‐human brain) / Quantitative analysis / Staining and Labeling - methods / synchrotron‐based X‐ray microscopy / Temperature / Tissues / X-rays