Bonthius Daniel J, McKim Ross, Koele Lindsey, Harb Harb, Karacay Bahri, Mahoney Jo, Pantazis Nicholas J
Department of Pediatrics, University of Iowa, Iowa City, IA 52242, USA.
Brain Res Brain Res Protoc. 2004 Nov;14(1):45-57. doi: 10.1016/j.brainresprot.2004.09.003.
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 multiple brain regions from a single set of sections.
体视学是对中枢神经系统各亚区神经元进行定量分析的重要技术。一种常用的体视学方法依赖于将组织包埋在甲基丙烯酸乙二醇酯中，以便制作出厚度不易收缩的切片。然而，使用甲基丙烯酸乙二醇酯进行体视学分析有几个缺点，包括对可处理组织大小的严格限制以及浸润过程通常需要较长时间。我们描述了一种利用冷冻状态下切割的组织切片的新型体视学方法。这种新方法依赖于将切片作为自由漂浮切片进行染色，并将这些切片用基于水的封片剂安装在载玻片上。通过这些方法处理的冷冻状态下切割的切片厚度几乎没有收缩或完全没有收缩，非常适合使用体视学的光学分割器或光学分数器方法进行体视学细胞计数。我们证明冷冻切片可用于高精度且低误差系数地估计神经元数量。由于可以将大的组织块切成冷冻切片，这种方法扩大了可有效用于体视学分析的组织范围，并能轻松地从同一组织切片中对多个脑区的神经元进行定量。我们应用这种新方法来估计10日龄小鼠新皮层和海马体中的神经元数量。该方法对于估计大的、细胞分布稀疏的细胞群体（如新皮层）以及小的、细胞密集的细胞（如齿状回颗粒细胞）都很有用。因此，与使用甲基丙烯酸乙二醇酯包埋进行体视学分析相比，冷冻切片方法具有许多潜在优势。这些优势包括可处理组织块大小的扩大、时间和成本的降低以及能够从一组切片中对多个脑区进行定量。
