Use of frozen sections to determine neuronal number in the murine hippocampus and neocortex using the optical disector and optical fractionator
Use of frozen sections to determine neuronal number in the murine hippocampus and neocortex using the optical disector and optical fractionator
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DOI：
10.1016/j.brainresprot.2004.09.003
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发表时间：
2004-11-01
期刊：
影响因子：
--
通讯作者：
Pantazis, NJ
中科院分区：
文献类型：
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作者：
Bonthius, DJ;McKim, R;Pantazis, NJ
体视学是一种重要的技术，用于定量的神经元在亚区域的中枢神经系统。常用的体视学方法依赖于将组织包埋在乙二醇甲基丙烯酸酯中，以允许产生抗厚度收缩的切片。然而，使用二醇甲基丙烯酸酯进行体视学具有若干缺点，包括对可处理的组织的尺寸的严格限制和渗透通常需要的长持续时间。我们描述了一种新的体视学方法，利用组织切片在冷冻状态下切割。这种新方法依赖于将切片染色为自由漂浮切片，并使用水基封固剂将这些切片封固到载玻片上。在冷冻状态下切割并通过这些方法处理的切片在厚度上经历很少或没有收缩，并且对于利用体视学的光学分割器或光学分级器方法进行体视学细胞计数是理想的。我们证明，冷冻切片可以用来估计神经元的数量与高精度和低误差系数。因为大的组织块可以被切割成冷冻切片，所以该方法扩大了可以被有效地处理用于体视学的组织的范围，并且容易地允许从来自相同组织切片的多个脑区域定量神经元。我们应用这种新的方法来估计10天大的小鼠的新皮层和海马中的神经元数量。该方法是有用的估计大，稀疏包装的细胞群体，如新皮质，和小，密集包装的细胞，如齿状回颗粒细胞。因此，冷冻切片方法提供了许多潜在的优势，使用乙二醇甲基丙烯酸酯嵌入体视学。这些优点包括可以处理的组织块的尺寸的扩展，所花费的时间和成本的减少，以及从单组切片量化多个脑区域的能力。(C)2004 Elsevier B.V.保留所有权利。
Stereology is an important technique for the quantification of neurons in subregions of the central nervous system. A commonly used method of stereology relies upon embedment of tissue in glycol methacrylates to allow production of sections that are resistant to shrinkage in thickness. However, the use of glycol methacrylates for stereology has several disadvantages, including severe constraints on the size of tissue that can be processed and the long duration of time often required for infiltration. We describe a novel method of stereology utilizing tissue sections cut in the frozen state. This new methodology relies upon the staining of sections as free-floating sections and upon the mounting of these sections onto slides with a water-based mounting media. Sections cut in the frozen state and processed by these methods undergo little or no shrinkage in thickness and are ideal for stereological cell counts utilizing either the optical disector or optical fractionator methods of stereology. We demonstrate that frozen sections can be utilized to estimate neuronal number with high degrees of precision and with low coefficients of error. Because large tissue blocks can be cut as frozen sections, this method expands the range of tissues that can be processed efficiently for stereology and readily allows quantification of neurons from multiple brain regions from the same tissue sections. We applied this new methodology to estimate neuronal numbers in the neocortex and hippocampus of 10-day-old mice. The method was useful for estimation of both large, sparsely packed cell populations, such as the neocortex, and small, densely packed cells, such as the dentate gyrus granule cells. Thus, frozen section methodology offers many potential advantages over the use of glycol methacrylate embedment for stereology. These advantages include expansion of the size of tissue blocks that can be processed, reduction in expended time and costs, and ability to quantify mulitiple brain regions from a single set of sections. (C) 2004 Elsevier B.V. All rights reserved.