Department of Genetics, Microbiology and Statistics, Faculty of Biology, University of Barcelona, Catalunya, Spain.
Institute of Biomedicine of the University of Barcelona (IBUB), Catalunya, Spain.
Methods Mol Biol. 2023;2680:81-91. doi: 10.1007/978-1-0716-3275-8_5.
Whole-mount in situ hybridization (WISH) is an extremely useful technique for visualizing specific mRNA targets and solving many biological questions. In planarians, this method is really valuable, for example, for determining gene expression profiles during whole-body regeneration and analyzing the effects of silencing any gene to determine their functions. In this chapter, we present in detail the WISH protocol routinely used in our lab, using a digoxigenin-labelled RNA probe and developing with NBT-BCIP. This protocol is basically that already described in Currie et al. (EvoDevo 7:7, 2016), which put together several modifications developed from several laboratories in recent years that improved the original protocol developed in the laboratory of Kiyokazu Agata in 1997. Although this protocol, or slight modifications of it, is the most common protocol in the planarian field for NBT-BCIP WISH, our results show that key steps such as the use and time of NAC treatment to remove the mucus need to be taken into account depending on the nature of the gene analyzed, especially for the epidermal markers.
整体原位杂交（WISH）是一种非常有用的技术，可用于可视化特定的 mRNA 靶标，并解决许多生物学问题。在扁形动物中，这种方法非常有价值，例如，可用于确定整个身体再生过程中的基因表达谱，并分析沉默任何基因的影响，以确定它们的功能。在本章中，我们详细介绍了我们实验室常规使用的 WISH 方案，该方案使用地高辛标记的 RNA 探针，并使用 NBT-BCIP 进行显色。该方案基本上是已经在 Currie 等人中描述的方案（EvoDevo 7:7, 2016），该方案结合了近年来来自多个实验室的几项改进，改进了 1997 年 Kiyokazu Agata 实验室开发的原始方案。尽管该方案或其轻微修改是扁形动物领域中用于 NBT-BCIP WISH 的最常见方案，但我们的结果表明，需要根据所分析基因的性质考虑一些关键步骤，例如 NAC 处理的使用和时间，以去除粘液，特别是对于表皮标记物。
