application of immunohistochemistry in stereology for quantitative assessment of neural cell populations illustrated in the g？ttingen minipig应用免疫组织化学的体视学定量评估的神经细胞群g插图业务minipig.pdf

Application of Immunohistochemistry in Stereology for Quantitative Assessment of Neural Cell Populations ¨ Illustrated in the Gottingen Minipig Jack Hou*, Jesper Riise, Bente Pakkenberg Research Laboratory for Stereology and Neuroscience, Copenhagen University Hospital, Bispebjerg, Denmark Abstract Background: 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 Gottingen 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 sh