Blach Patricia, Keskin Sercan, de Jonge Niels
INM-Leibniz Institute for New Materials; Department of Physics, Saarland University.
INM-Leibniz Institute for New Materials.
J Vis Exp. 2020 Sep 21(163). doi: 10.3791/61458.
A protocol is described for investigating the human epidermal growth factor receptor 2 (HER2) in the intact plasma membrane of breast cancer cells using scanning transmission electron microscopy (STEM). Cells of the mammalian breast cancer cell line SKBR3 were grown on silicon microchips with silicon nitride (SiN) windows. Cells were chemically fixed, and HER2 proteins were labeled with quantum dot nanoparticles (QDs), using a two-step biotin-streptavidin binding protocol. The cells were coated with multilayer graphene to maintain a hydrated state, and to protect them from electron beam damage during STEM. To examine the stability of the samples under electron beam irradiation, a dose series experiment was performed. Graphene-coated and non-coated samples were compared. Beam induced damage, in the form of bright artifacts, appeared for some non-coated samples at increased electron dose D, while no artifacts appeared on coated samples.
本文描述了一种使用扫描透射电子显微镜（STEM）研究乳腺癌细胞完整质膜中人类表皮生长因子受体2（HER2）的实验方案。哺乳动物乳腺癌细胞系SKBR3的细胞在带有氮化硅（SiN）窗口的硅微芯片上生长。细胞经过化学固定，采用两步生物素-链霉亲和素结合方案，用量子点纳米颗粒（QDs）标记HER2蛋白。细胞用多层石墨烯包覆以保持水合状态，并在STEM过程中保护它们免受电子束损伤。为了研究样品在电子束照射下的稳定性，进行了剂量系列实验。比较了石墨烯包覆和未包覆的样品。在增加电子剂量D时，一些未包覆的样品出现了以明亮伪像形式存在的束致损伤，而包覆的样品上未出现伪像。
