A simple protocol for paraffin-embedded myelin sheath staining with osmium tetroxide for light microscope observation
A simple protocol for paraffin-embedded myelin sheath staining with osmium tetroxide for light microscope observation
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DOI：
10.1002/jemt.20577
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发表时间：
2008-07-01
影响因子：
2.5
通讯作者：
Geuna, Stefano
中科院分区：
文献类型：
--
作者：
Di Scipio, Federica;Raimondo, Stefania;Geuna, Stefano
周围神经纤维再生的实验研究越来越受到基础和临床研究者的重视。有髓神经纤维形态学评价是周围神经再生研究的重要内容。有髓神经纤维光学显微镜成像的金标准是树脂包埋半薄切片的甲苯胺蓝染色。然而，许多研究人员不知道，这种方法产生的髓鞘深染是由于四氧化锇后固定，而不是由于甲苯胺蓝。在这篇文章中，我们描述了一个简单的预包埋协议染色髓鞘石蜡包埋的神经标本使用四氧化锇。标本经多聚甲醛固定后，用2%四氧化锇浸泡2h，常规脱水，石蜡包埋。然后可以直接在显微镜下观察切片或使用常规组织学方法复染。特别好的结果，获得了与马森的三色复染，它允许在甲苯胺蓝染色的树脂切片中检测不到的神经中的结缔结构的成像。最后，我们描述了一个简单的协议，锇蚀刻的部分，这使得进一步的免疫组化分析可能在相同的标本。总之，我们的结果表明，本文中描述的协议是一个有效的替代传统的树脂包埋为基础的协议：它是便宜得多，可以通过任何组织学实验室，并允许进行免疫组织化学分析。
Experimental investigation of peripheral nerve fiber regeneration is attracting more and more attention among both basic and clinical researchers. Assessment of myelinated nerve fiber morphology is a pillar of peripheral nerve regeneration research. The gold standard for light microscopic imaging of myelinated nerve fibers is toluidine blue staining of resin-embedded semithin sections. However, many researchers are unaware that the dark staining of myelin sheaths typically produced by this procedure is due to osmium tetroxide postfixation and not due to toluidine blue. In this article, we describe a simple pre-embedding protocol for staining myelin sheaths in paraffin-embedded nerve specimens using osmium tetroxide. The method involves immersing the specimen in 2% osmium tetroxide for 2 h after paraformaldeyde fixation, followed by routine dehydration and paraffin embedding. Sections can then be observed directly under the microscope or counterstained using routine histological methods. Particularly good results were obtained with Masson's trichrome counterstain, which permits the imaging of connective structures in nerves that are not detectable in toluidine blue-stained resin sections. Finally, we describe a simple protocol for osmium etching of sections, which makes further immunohistochemical analysis possible on the same specimens. Taken together, our results suggest that the protocol described in this article is a valid alternative to the conventional resin embedding-based protocol: it is much cheaper, can be adopted by any histological laboratory, and allows immunohistochemical analysis to be conducted.