Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany.
Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany.
Methods Mol Biol. 2023;2680:121-155. doi: 10.1007/978-1-0716-3275-8_8.
High-content fluorescence microscopy combines the efficiency of high-throughput techniques with the ability to extract quantitative information from biological systems. Here we describe a modular collection of assays adapted for fixed planarian cells that enable multiplexed measurements of biomarkers in microwell plates. These include protocols for RNA fluorescent in situ hybridization (RNA FISH) as well as immunocytochemical protocols for quantifying proliferating cells targeting phosphorylated histone H3 as well as 5-bromo-2'-deoxyuridine (BrdU) incorporated into the nuclear DNA. The assays are compatible with planarians of virtually any size, as the tissue is disaggregated into a single-cell suspension before fixation and staining. By sharing many reagents with established planarian whole-mount staining protocols, preparation of samples for high-content microscopy adoption requires little additional investment.
高内涵荧光显微镜将高通量技术的效率与从生物系统中提取定量信息的能力相结合。在这里，我们描述了一组适用于固定涡虫细胞的模块化检测方法，这些方法可实现微孔板中生物标志物的多重测量。其中包括用于 RNA 荧光原位杂交 (RNA FISH) 的方案，以及用于定量检测磷酸化组蛋白 H3 以及掺入核 DNA 的 5-溴-2'-脱氧尿苷 (BrdU) 的免疫细胞化学方案。这些检测方法与几乎任何大小的涡虫都兼容，因为组织在固定和染色之前被分散成单细胞悬浮液。由于与已建立的涡虫全组织染色方案共享许多试剂，因此采用高内涵显微镜制备样品几乎不需要额外的投资。
