Brotchie D, Birch M, Roberts N, Howard C V, Smith V A, Grierson I
Unit of Ophthalmology, Department of Medicine, University of Liverpool, UK.
J Neurosci Methods. 1999 Feb 1;87(1):77-85. doi: 10.1016/s0165-0270(98)00168-x.
The use of picrosirius red to localise connective tissue in thin tissue sections viewed by bright-field microscopy is well documented. Its use on thin tissue sections imaged by fluorescence confocal microscopy has also been reported. Here we describe modifications to published procedures that allow picrosirius red staining of thick 60-microm sections and their subsequent analysis by confocal microscopy. The use of phosphomolybdic acid pre-treatment was found to be essential for confocal analysis; in addition to preventing non-specific staining, it also quenched tissue autofluorescence. By incubating sections free-floating, pre-treating them with phosphomolybdic acid for 30 min and imaging them using an argon ion laser we were able to use confocal microscopy to image the entire depth of 60-microm human optic nerve and nerve head sections stained with picrosirius red. The application of this modified picrosirius red and confocal microscopy technique should be useful for analysing the three-dimensional structure of the optic nerve and other tissues with a similarly complex arrangement of connective tissue.
在明场显微镜下观察薄组织切片中使用天狼星红对结缔组织进行定位的方法已有充分记录。也有报道称其用于荧光共聚焦显微镜成像的薄组织切片。在此，我们描述了对已发表程序的改进，这些改进使得能够对60微米厚的切片进行天狼星红染色，并随后通过共聚焦显微镜进行分析。发现使用磷钼酸预处理对于共聚焦分析至关重要；除了防止非特异性染色外，它还能淬灭组织自发荧光。通过将切片进行游离孵育，用磷钼酸预处理30分钟，并使用氩离子激光对其成像，我们能够利用共聚焦显微镜对用天狼星红染色的60微米厚的人视神经和神经头切片的整个深度进行成像。这种改良的天狼星红和共聚焦显微镜技术的应用对于分析视神经和其他具有类似复杂结缔组织排列的组织的三维结构应该是有用的。
