IG-TEM 这么繁琐吗
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). As controls, some of the grids were not exposed to the primary antibody. 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. 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.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,一些网格没有暴露于一抗。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。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。
求做过的大神来指点下~~:loveliness: 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
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。 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 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:curse:
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