Improving structural integrity of cryosections for immunogold labeling
Improving structural integrity of cryosections for immunogold labeling
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DOI：
10.1007/bf02473201
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发表时间：
1996-07-01
影响因子：
2.3
通讯作者：
Slot, JW
中科院分区：
文献类型：
--
作者：
Liou, W;Geuze, HJ;Slot, JW
根据Tokuyasu制备的冷冻切片固定材料是免疫细胞化学的良好底物。然而，结构缺陷的出现限制了这种方法的分辨率。我们发现，切片解冻并从冷冻室转移到EM网格上的支撑膜的步骤对于结构保存是最关键的。在解冻过程中，切片在其上展开的转移介质的表面张力很容易因过度拉伸而损坏其结构。通过用甲基纤维素和蔗糖的混合物代替传统的蔗糖转移介质，我们能够减轻过度拉伸的问题，从而大大提高了薄冷冻切片的结构完整性。此外，解冻后从切片中提取材料会导致结构损坏，特别是在交联不足时。在转移介质中掺入乙酸双氧铀可以进一步帮助在免疫标记过程中保持切片的结构完整性。在甲基纤维素/醋酸铀混合物中直接取出并干燥的切片具有良好的超微结构。这样的准备工作可以提供新的洞察亚细胞的细节，是一个有效的备份免疫标记的部分，就其形态。新鲜冷冻组织的冰冻切片可以通过使用含有固定剂的转移培养基进行保存以用于免疫标记。如果认为组织的化学固定会诱导形态学伪影或抗原再分布，则这种方法可能具有优势。
Cryosections of aldehyde-fixed material prepared according to Tokuyasu are a good substrate for immunocytochemistry. However, structural defects occur that limit the resolution of this approach. We found that the step during which sections are thawed and transferred from the cryochamber to the supporting film on an EM grid is most critical for structural preservation. Surface tension of the transfer medium, on which sections are spread during thawing, can easily damage their structure by overstretching. By substituting a mixture of methylcellulose and sucrose for the conventional sucrose transfer medium, we were able to alleviate the problem of overstretching, thus improving greatly the structural integrity of thin cryosections. Also, material extraction from the sections after thawing causes structural damage, particularly when cross-linking is deficient. Incorporation of uranyl acetate in the transfer medium can then further help to maintain the structural integrity of the sections during the immunolabeling procedure. Excellent ultrastructure was featured in sections picked up and dried directly in methylcellulose/uranyl acetate mixtures. Such preparations can provide new insight into subcellular details and is an efficient back-up for immunolabeled sections in respect of their morphology. Cryosections from fresh frozen tissue can be preserved for immunolabeling by using transfer media that contain fixatives. This approach may have advantages if chemical fixation of tissue is thought to induce morphological artifacts or antigen redistribution.