Research Laboratory for Stereology and Neuroscience, Copenhagen University Hospital, Bispebjerg, Denmark.
PLoS One. 2012;7(8):e43556. doi: 10.1371/journal.pone.0043556. Epub 2012 Aug 15.
Stereology is the study of estimating geometric quantities. When successfully applied, the combination of immunohistochemistry (IHC) and stereology eliminates intra- and interobserver variability for cell type identification.
METHODOLOGY/PRINCIPAL FINDINGS: We propose a method to validate existing antibody based cell type markers for stereological application. Comparison was made on the 100-days-old Göttingen minipig (G-mini) neocortex between estimates of total neuron number derived from Giemsa staining using morphological criteria and immunohistochemistry-based cell counting with NeuN. The mean total neuron numbers estimated by the two staining methods were not significantly different. Estimated quantities, including glial cell number, neocortical volume, cell densities and glial-to-neuron ratio were also presented. Additionally, we assessed other commonly used glial markers and discussed how to evaluate the advantages and disadvantages of these markers for stereological estimation of cell number.
CONCLUSION/SIGNIFICANCE: The concordance in quantitative estimates of total neuron number derived from NeuN- and Giemsa-stained sections provides evidence for the sensitivity and specificity of NeuN as a neuronal marker in the G-mini. Although time-consuming, quantitative validation of IHC should always be considered in stereological studies if there is doubt of the sensitivity, specificity, or reproducibility of cell type markers. Inaccurate staining may cause both over- and underestimation of the total cell number and inflict considerable limitation when analyzing the results.
体视学是研究估计几何量的学科。当成功应用时，免疫组织化学（IHC）和体视学的结合消除了细胞类型识别的观察者内和观察者间的变异性。
方法/主要发现：我们提出了一种验证现有的基于抗体的细胞类型标志物用于体视学应用的方法。在 100 天大的哥廷根小型猪（G-mini）新皮质中，我们比较了基于形态学标准的 Giemsa 染色和基于 NeuN 的免疫组织化学细胞计数对总神经元数量的估计。两种染色方法估计的总神经元数量没有显著差异。还提供了包括神经胶质细胞数量、新皮质体积、细胞密度和神经胶质细胞与神经元比率在内的估计数量。此外，我们评估了其他常用的神经胶质标志物，并讨论了如何评估这些标志物用于细胞数量体视学估计的优缺点。
结论/意义：来自 NeuN 和 Giemsa 染色切片的总神经元数量定量估计的一致性为 NeuN 作为 G-mini 神经元标志物的敏感性和特异性提供了证据。尽管耗时，但如果对细胞类型标志物的敏感性、特异性或可重复性有疑问，在体视学研究中始终应考虑对 IHC 进行定量验证。不准确的染色可能导致总细胞数量的高估和低估，并在分析结果时造成相当大的限制。
