Improved methods for ultracryotomy of CNS tissue for ultrastructural and immunogold analyses
Improved methods for ultracryotomy of CNS tissue for ultrastructural and immunogold analyses
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DOI：
10.1016/j.jneumeth.2005.11.007
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发表时间：
2006-06-15
影响因子：
3
通讯作者：
Tohyama, Koujiro
中科院分区：
文献类型：
--
作者：
Akagi, Takumi;Ishida, Kinji;Tohyama, Koujiro
我们检查了用于中枢神经系统组织的超低温冷冻术方案的每个步骤，以确定和克服一些方法上的困难。以下三个步骤对该方法的成功至关重要：（1）在使用前立即对网格进行预处理，使其具有亲水性;（2）在超低温冷冻切片过程中仔细收集冷冻切片;（3）在用PVA-UA染色后干燥之前，去除适量的过量聚（乙烯醇）-醋酸铀酰（PVA-UA）。通过考虑上述三个关键步骤，我们成功地获得了超薄冷冻切片，包括超微结构保存良好的连续切片，以及用于评价样品质量和选择感兴趣区域进行超微结构分析的半薄冷冻切片。突触和有髓纤维的精细结构保存完好。星形胶质细胞谷氨酸转运体（GLAST）、代谢型谷氨酸受体1（mGluR 1）和神经丝蛋白免疫染色后的金颗粒定位与以往报道一致，所有病例的超微结构均保存良好，这些发现将有助于研究人员对神经组织冰冻切片进行超微结构和免疫金分析。(c)2005 Elsevier B. V.保留所有权利。
We examined each step of the protocol for ultracryotomy for central nervous system tissue in order to define and overcome some of the methodological difficulties. The following three steps emerged as critical for the method's success: (1) pretreatment of grids to render them hydrophilic immediately before use; (2) careful collection of ultrathin cryosections during ultracryotomy; (3) removal of the appropriate amount of excess poly(vinyl alcohol)-uranyl acetate (PVA-UA) prior to drying after staining with PVA-UA. By taking account of the three critical steps described above, we succeeded in obtaining ultrathin cryosections, including serial sections, with excellent preservation of ultrastructure, as well as semithin cryosections which are useful for evaluating the quality of the samples and for selecting areas of interest for ultrastructural analysis.Cytoplasmic organelles in neurons and glial cells, and the fine structure of synapses and myelinated fibers were well preserved. The localization of gold particles after immunostaining for astrocytic glutamate transporter (GLAST), metabotropic glutamate receptor 1 (mGluR1) and neurofilament protein was consistent with previous reports and ultrastructure was well-preserved in all cases.These findings should be helpful to researchers wishing to carry out ultrastructural and immunogold analyses of cryosections of nervous tissue. (c) 2005 Elsevier B.V. All rights reserved.