Schmitz C, Dafotakis M, Heinsen H, Mugrauer K, Niesel A, Popken G J, Stephan M, Van de Berg W D, von Hörsten S, Korr H
Department of Anatomy and Cell Biology, RWTH University of Aachen, Pauwelsstrasse/Wendlingweg 2, 52057, Aachen, Germany.
J Chem Neuroanat. 2000 Oct;20(1):21-9. doi: 10.1016/s0891-0618(00)00075-2.
Adequate tissue preparation is essential for both modern stereological and immunohistochemical investigations. However, combining these methodologies in a single study presents a number of obstacles pertaining to optimal histological preparation. Tissue shrinkage and loss of nuclei/nucleoli from the unprotected section surfaces of unembedded tissue used for immunohistochemistry may be problematic with regard to adequate stereological design. In this study, frozen cryostat sections from hippocampal and cerebellar regions of two rat strains and cerebellar and cerebral regions from a human brain were analyzed to determine the potential impact of these factors on estimates of neuron number obtained using the optical disector. Neuronal nuclei and nucleoli were clearly present in thin sections of snap-frozen rat (3 microm) and human (6 microm) tissue, indicating that neuronal nuclei/nucleoli are not unavoidably lost from unprotected section surfaces of unembedded tissue. In order to quantify the potential impact of any nuclear loss, optical fractionator estimates of rat hippocampal pyramidal cells in areas CA1-3 and cerebellar granule and Purkinje cells were made using minimal (1 microm) upper guard zones. Estimates did not differ from data reported previously in the literature. This data indicates that cryostat sections of snap-frozen nervous tissue may successfully be used for estimating total neuronal numbers using optical disectors.
对于现代体视学和免疫组织化学研究而言，充分的组织制备至关重要。然而，在一项研究中结合这些方法存在一些与最佳组织学制备相关的障碍。对于适当的体视学设计而言，用于免疫组织化学的未包埋组织的未受保护切片表面出现组织收缩以及细胞核/核仁丢失可能会成为问题。在本研究中，分析了来自两种大鼠品系海马和小脑区域以及人脑小脑和大脑区域的冷冻恒冷箱切片，以确定这些因素对使用光学分割器获得的神经元数量估计值的潜在影响。在速冻大鼠（3微米）和人类（6微米）组织的薄片中，神经元细胞核和核仁清晰可见，这表明神经元细胞核/核仁并非不可避免地从未包埋组织的未受保护切片表面丢失。为了量化任何细胞核丢失的潜在影响，使用最小（1微米）上保护带对大鼠海马CA1 - 3区的锥体细胞以及小脑颗粒细胞和浦肯野细胞进行了光学分数估计。估计值与先前文献报道的数据没有差异。该数据表明，速冻神经组织的恒冷箱切片可成功用于使用光学分割器估计神经元总数。
