Lowry A, Wilcox D, Masson E A, Williams P E
Department of Biological Sciences, University of Hull, UK.
J Anat. 1997 Oct;191 ( Pt 3)(Pt 3):367-74. doi: 10.1046/j.1469-7580.1997.19130367.x.
Methods are described for the semiquantitative analysis of the connective tissue components of human peripheral nerve using light microscopy. General histological preservation was assessed using haematoxylin and eosin staining and the distribution of collagen type IV was investigated using immunohistochemistry. Several techniques were investigated to establish the one giving optimum structural preservation, immunobinding and greatest contrast for image analysis. Frozen sections were unsuitable for this tissue and paraffin wax sections were therefore used. Alcohol fixation was rejected due to poor preservation of the endoneurium, although immunobinding was excellent. Ice-cold formalin fixation for 24 h was found to be adequate for structural preservation and antibody binding, provided that a protease step was introduced. Trypsin was found to be superior to pepsin for exposing collagen type IV epitopes. Of the detection systems investigated indirect immunofluorescence was not suitable due to considerable autofluorescence of the nerve. The avidin-biotin method provided the greatest contrast, and was therefore the detection method of choice for image analysis. The optimum techniques for image analysis were then used on control human sural nerve to ascertain the best comparative method for collagen type IV in the perineurium. A method of semiquantitative analysis is described which takes into account the fact that there is a close linear relationship between collagen content per unit of perineurium and perineurial perimeter as fascicle size increases in peripheral nerve. This means that data from 2 different sample groups can easily be compared, provided that a range of fascicle sizes is analysed in each case.
描述了使用光学显微镜对人周围神经结缔组织成分进行半定量分析的方法。使用苏木精和伊红染色评估一般组织学保存情况，并使用免疫组织化学研究IV型胶原的分布。研究了几种技术，以确定能实现最佳结构保存、免疫结合以及图像分析最大对比度的技术。冷冻切片不适用于该组织，因此使用石蜡切片。由于神经内膜保存不佳，酒精固定被排除，尽管免疫结合效果极佳。发现冰冰冷福尔马林固定24小时足以实现结构保存和抗体结合，前提是引入蛋白酶步骤。发现胰蛋白酶在暴露IV型胶原表位方面优于胃蛋白酶。在研究的检测系统中，间接免疫荧光由于神经有相当大的自发荧光而不适用。抗生物素蛋白-生物素方法提供了最大的对比度，因此是图像分析的首选检测方法。然后将图像分析的最佳技术应用于对照人腓肠神经，以确定神经束膜中IV型胶原的最佳比较方法。描述了一种半定量分析方法，该方法考虑到随着周围神经中神经束大小增加，单位神经束膜胶原含量与神经束膜周长之间存在密切的线性关系。这意味着，只要在每种情况下分析一系列神经束大小，就可以轻松比较来自2个不同样本组的数据。
