Pushie M J, Hollings A, Reinhardt J, Webb S M, Lam V, Takechi R, Mamo J C, Paterson P G, Kelly M E, George G N, Pickering I J, Hackett M J
Department of Surgery, Division of Neurosurgery, College of Medicine, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan S7N 5E5, Canada.
Curtin Health Innovation Research Institute, Curtin University, Perth, WA 6102, AUS.
J Anal At Spectrom. 2020 Nov 1;35(11):2498-2508. doi: 10.1039/d0ja00323a. Epub 2020 Aug 19.
Transition metal ions (Fe, Mn, Cu, Zn) are essential for healthy brain function, but altered concentration, distribution, or chemical form of the metal ions has been implicated in numerous brain pathologies. Currently, it is not possible to image the cellular or sub-cellular distribution of metal ions and therefore, studying brain-metal homeostasis largely relies on elemental mapping. Sample preparation methods that accurately preserve the elemental distribution are essential if one wishes to translate the knowledge of elemental distributions measured toward increased understanding of chemical and physiological pathways of brain disease. The choice of sample preparation is particularly important for metal ions that exist in a labile or mobile form, for which the distribution could be easily distorted by inappropriate sample preparation. One of the most widely studied brain structures, the hippocampus, contains a large pool of labile and mobile Zn. Herein, we describe how sucrose cryoprotection, the gold standard method of preparing tissues for immuno-histochemistry or immuno-fluorescence, which is also often used as a sample preparation method for elemental mapping studies, drastically alters hippocampal Zn distribution. Based on the results of this study, in combination with a comparison against the strong body of published literature that has used either rapid plunge freezing of brain tissue, or sucrose cryo-protection, we strongly urge investigators in the future to cease using sucrose cryoprotection as a method of sample preparation for elemental mapping, especially if Zn is an analyte of interest.
过渡金属离子（铁、锰、铜、锌）对大脑的健康功能至关重要，但金属离子浓度、分布或化学形式的改变与多种脑部病变有关。目前，尚无法对金属离子的细胞或亚细胞分布进行成像，因此，研究大脑金属稳态在很大程度上依赖于元素图谱分析。如果希望将所测得的元素分布知识转化为对脑部疾病化学和生理途径的更多了解，那么能够准确保留元素分布的样品制备方法至关重要。对于以不稳定或可移动形式存在的金属离子，样品制备的选择尤为重要，因为不适当的样品制备可能会轻易扭曲其分布。海马体是研究最为广泛的脑结构之一，其中含有大量不稳定且可移动的锌。在此，我们描述了蔗糖冷冻保护法，这一用于免疫组织化学或免疫荧光组织制备的金标准方法，同时也常用于元素图谱分析研究的样品制备，它是如何极大地改变海马体锌分布的。基于本研究结果，并与大量已发表文献进行比较，这些文献要么采用脑组织快速冷冻法，要么采用蔗糖冷冻保护法，我们强烈敦促未来的研究人员停止使用蔗糖冷冻保护法作为元素图谱分析的样品制备方法，尤其是当锌是感兴趣的分析物时。
