A simple and rapid method for combining fluorescent in situ RNA hybridization (FISH) and immunofluorescence in the C. elegans germline.
A simple and rapid method for combining fluorescent in situ RNA hybridization (FISH) and immunofluorescence in the C. elegans germline.
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作者：
Yoon DS;Pendergrass DL;Lee MH
特定组织中 RNA 和蛋白质的成像为了解发育过程中的基因表达开辟了充足的途径。最近，开发了一种荧光原位RNA杂交（FISH）方法来分析内源mRNA的时空表达模式。然而，将 FISH 与免疫荧光结合起来具有挑战性，因为两种方法的反应条件在多种方面存在冲突。在本报告中，我们开发了一种简单快速的方法来检测 RNA 和相关蛋白，更好地保存线虫种系的精细结构。该方法将为同一种系中RNA及其相关蛋白的体内成像提供新工具，这也使得体内细胞水平的RNA/蛋白质复合物同时可视化成为可能。开发一种简单快速的 FISH 方法，可以更好地保存精细结构。将 FISH 与免疫荧光结合用于秀丽隐杆线虫种系。标记挤出的性腺，而不是整个蠕虫，以防止非特异性体细胞自发荧光。
Imaging of RNAs and proteins in specific tissues has opened ample avenues to understand gene expression during development. Recently, a fluorescent in situ RNA hybridization (FISH) method has been developed to analyze the spatio-temporal expression patterns of endogenous mRNAs. However, combining FISH with immunofluorescence is challenging as the reaction conditions for the two procedures conflict in multiple ways. In this report, we developed a simple and rapid method to detect both RNAs and associated proteins with better preservation of the fine structure in the C. elegans germline. This method will provide new tools for in vivo imaging of RNAs and their associated proteins in the same germline, which also enables simultaneous visualization of RNA/protein complex at the cellular level in vivo. Developing a simple and rapid FISH method with better preservation of the fine structure. Combining FISH with immunofluorescence in C. elegans germline. Labeling extruded gonads, instead of the whole worms, to prevent non-specific somatic autofluorescence.