Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada.
Donnelly Centre, University of Toronto, Toronto, ON M5S 3E1, Canada.
STAR Protoc. 2021 Jun 28;2(3):100632. doi: 10.1016/j.xpro.2021.100632. eCollection 2021 Sep 17.
Automated high-content immunofluorescence (IF) microscopy is used to monitor and quantify localization of the TGFβ/Smads and Taz/Yap Hippo effectors in mouse epithelial EpH4 cells transfected with Taz/Yap siRNAs. The nuclear-to-cytoplasmic protein ratios obtained by IF are converted into normalized masses by estimating the ratio of the compartment volumes. This method has the advantage that endogenous rather than tagged proteins are tracked and that knockdown of Taz/Yap can be simultaneously monitored at the single-cell level. For complete details on the use and execution of this protocol, please refer to Labibi et al. (2020).
自动化高通量免疫荧光（IF）显微镜用于监测和定量分析 TGFβ/Smads 和 Taz/Yap Hippo 效应物在转染 Taz/Yap siRNA 的小鼠上皮细胞 EpH4 中的定位。通过 IF 获得的核质蛋白比通过估计隔室体积比转化为归一化质量。这种方法的优点是可以跟踪内源性而不是标记蛋白，并且可以在单细胞水平同时监测 Taz/Yap 的敲低情况。有关该方案使用和执行的完整详细信息，请参阅 Labibi 等人。（2020）。
