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: IG-TEM 这么繁琐吗 [打印本页]
作者: liuhaoisboy    时间: 2015-12-9 09:16
标题: IG-TEM 这么繁琐吗
[backcolor=rgba(255, 255, 255, 0.8)]Fixed specimens at an optimal concentration were placed onto a 400-mesh carbon/formvar coated grids and allowed to absorb to the formvar for a minimum of 1 min. For immunogold staining the grids were placed into a blocking buffer for a block/permeabilization step for 1 h. Without rinsing, the grids were immediately placed into the primary antibody at the appropriate dilution overnight at 4 °C (1:300 anti-CD9 ab92726, Abcam and anti-GPC1 PIPA528055, Thermo Scientific). [backcolor=rgba(255, 255, 255, 0.8)]As controls, some of the grids were not exposed to the primary antibody. [backcolor=rgba(255, 255, 255, 0.8)]The next day, all the grids were rinsed with PBS then floated on drops of the appropriate secondary antibody attached with 10-nm gold particles (AURION) for 2 h at room temperature. [backcolor=rgba(255, 255, 255, 0.8)]Grids were rinsed with PBS and were placed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 15 min. After rinsing in PBS and distilled water the grids were allowed to dry and stained for contrast using uranyl acetate.[backcolor=rgba(255, 255, 255, 0.8)]

固定标本的最佳浓度被放置到一个400 -网状碳/ formvar涂层网格和允许吸收formvar染色然后至少1分钟。为电镜观察,网格被放到一个阻塞缓冲块/透化作用的步骤1 h。无需冲洗,网是立即放入一抗在适当的稀释在4°C过夜(anti-CD9 ab92726,Abcam和anti-GPC1 PIPA528055,热科学)。作为对照[backcolor=rgba(255, 255, 255, 0.8)],一些网格没有暴露于一抗。[backcolor=rgba(255, 255, 255, 0.8)]第二天，所有的网格用PBS冲洗，然后孵育适当的二次抗体滴附有soi黄金粒子在室温下(AURION)2h。[backcolor=rgba(255, 255, 255, 0.8)]网格冲洗PBS和放置在0.1 2.5%的戊二醛磷酸缓冲15分钟。在PBS和蒸馏水冲洗后网格被允许干和彩色对比使用醋酸双氧铀。

[backcolor=rgba(255, 255, 255, 0.8)]求做过的大神来指点下~~ 他这是因为要胶体金标记，所以才孵育抗体的吧？醋酸双氧铀负染一般不用一抗、二抗的步骤吧?正准备做，求大神指点


来源文献：
Glypican-1 identifies cancer exosomes and detects early pancreatic cancer .2015.nature

作者: hzangs    时间: 2015-12-9 10:20
如果只是打个负染电镜的话，很简单的。  但是免疫胶体金电镜 我们这边电镜室老师的话  ：那个难做
作者: liuhaoisboy    时间: 2015-12-10 11:15
hzangs 发表于 2015-12-9 10:20
如果只是打个负染电镜的话，很简单的。  但是免疫胶体金电镜 我们这边电镜室老师的话  ：那个难做 ...

恩。是的。文献中是用一抗、二抗的方法标记出来外泌体表面的CD9、CD81等表面标志分子。代替WB的检测。高大上
作者: hzangs    时间: 2015-12-11 10:38
liuhaoisboy 发表于 2015-12-10 11:15
恩。是的。文献中是用一抗、二抗的方法标记出来外泌体表面的CD9、CD81等表面标志分子。代替WB的检测。高 ...

免疫胶体金电镜的目的在于 标记膜泡表面的marker分子， 这样电镜的时候 是可以看到膜泡表面有胶体金颗粒附着。  一般来说只有电镜下exosome的形状不典型的时候 才会去做免疫胶体金。 目的在于证明电镜下观察到的不典型的膜泡结构是exosome。

试验中一般都是用简单实验来代替复杂实验， 你想想 免疫胶体金容易呢 还是 WB容易呢？  所以说，如果你的电镜结果是很典型的exosome 结构， 那基本上就不需要做胶体金电镜了。



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