外泌体之家 | 细胞外膜泡领域核心平台—exosomes & microvesicles—小膜泡大作用

标题: 求助外泌体的电镜处理方法 [打印本页]
作者: 龙吟风    时间: 2016-7-6 15:12
标题: 求助外泌体的电镜处理方法
学校电镜室按中外文献报道的方法均未拍摄出满意的电镜图片，求助外泌体的电镜处理方法，如果能给出出处就更感谢了。
作者: 龙吟风    时间: 2016-7-6 17:18
希望有经验的战友给予帮助
作者: 暂无昵称    时间: 2016-7-8 11:47
好歹把你用过的方法贴出来啊，直接一句中外文献都用了，你让其他人怎么给建议...
作者: hzangs    时间: 2016-7-8 11:47
http://www.exosome.com.cn/forum. ... 3D1&_dsign=032c61b6这个贴子里有
作者: 龙吟风    时间: 2016-7-8 14:48
暂无昵称 发表于 2016-7-8 11:47
好歹把你用过的方法贴出来啊，直接一句中外文献都用了，你让其他人怎么给建议... ...

疏忽了，谢谢。
作者: 龙吟风    时间: 2016-7-8 14:58
本帖最后由 龙吟风 于 2016-7-8 14:59 编辑

1．3．3透射电子显微镜观察血清外泌体形态
(1)取分装后的血清外泌体，解冻后重悬在200ul 1xPBS液中，混匀；
(2)取10ul外泌体悬液滴至电镜铜网状栅中，使悬液在铜网上保留至少20
分钟：
(3)1 xPBS液50ul固定1分钟后，重复2．3次；
(4)4％戊二醛10ul固定5分钟，滤纸吸干，lxPBS液洗涤1次，室温自然
晾干；
(5)将洗涤后的铜网安装在Hitachi．7500透射电子显微镜下观察并拍照。这是参考湘雅的一篇博士论文
作者: 龙吟风    时间: 2016-7-8 15:10
本帖最后由 龙吟风 于 2016-7-8 15:13 编辑

Transmission electron microscopy.
The samples were dissolved in HEPES (4-[2-hydroxyethyl]-1-piperazine ethanesulfonic acid) buffer, and a drop of the suspension was placed on a sheet of parafilm.
A carbon-coated copper grid was floated on the drop for 10 seconds. Then, the grid was removed, and excess liquid was drained by touching the edge of the grid against a piece of clean filter paper. The grid was touched onto a drop of 2% uranyl acetate or phosphotungstic acid, pH 7.0, for approximately 5 seconds, and excess liquid was drained off. The grid was allowed to dry for several minutes and then examined using a JEM-1200 EX microscope (JEOL,Akishima, Japan) at 80 kiloelectron volts.
Tanaka,Y，H．Kamohara,K．Kinoshita,et a1．Clinical impact of serum exosomal microRNA-2 1 as a clinical biomarker in human esophageal squamouscell carcinoma．[J]Cancer,201 3．1 1 9(6)：P．1 1 59—67．该种方法运用在多篇文章中
Transmission electron microscopy
TEM was performed by the method of Thery et al. [50].Exosomes isolated by ExoQuick reagent were suspended in PBS. A drop of this solution was allowed to settle on a goldcoated grid, fixed in 1 % glutaraldehyde, washed for 2 min in double-distilled water, and incubated in uranyl oxylate for 5 min, followed by incubation with three separate drops of methyl cellulose with uranyl acetate—5 min with the first two drops and 10 min with the last drop—and finally, methyl cellulose-uranyl acetate was removed by slow-drag on edge on filter paper. Exosomes were visualized by standard TEM with a Philips CM120 microscope
Munagala, R., F. Aqil and R.C. Gupta, Exosomal miRNAs as biomarkers of recurrent lung cancer. Tumor Biology, 2016.以上就是用过的方法，电镜室的老师说步骤不详细，很多没法做。
作者: 龙吟风    时间: 2016-7-12 15:11
hzangs 发表于 2016-7-8 11:47
http://www.exosome.com.cn/forum. ... 3D1&_dsign=032c61b6这个贴子里有

帖子看过，不知道您试过没有，靠不靠谱？
作者: 龙吟风    时间: 2016-7-12 15:12
求帮助
作者: hzangs    时间: 2016-7-12 15:18
龙吟风 发表于 2016-7-12 15:11
帖子看过，不知道您试过没有，靠不靠谱？

我在这个贴子里秀的电镜图 就是这么打出来的



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