Laboratoire Reproduction et Développement des Plantes, Ecole Normale Supérieure Lyon, Lyon, France.
1] Laboratoire Reproduction et Développement des Plantes, Ecole Normale Supérieure Lyon, Lyon, France. [2] Laboratoire Joliot Curie, Ecole Normale Supérieure Lyon, Lyon, France.
Nat Protoc. 2014 Oct;9(10):2464-75. doi: 10.1038/nprot.2014.162. Epub 2014 Sep 25.
In situ mRNA hybridization is one of the most powerful techniques for analyzing patterns of gene expression. However, its usefulness is limited in complex plant tissues by the need to fix, embed and section samples before localizing the desired mRNA. Here we present a robust whole-mount in situ hybridization method that allows easy access to patterns of gene expression in intact, complex tissues, such as the inflorescence apex of Arabidopsis thaliana. The tissue is first fixed and then permeabilized by treatment with a cocktail of cell wall-digesting enzymes that has been optimized to preserve the integrity of tissue structures, while also permitting the detection of expression patterns in deep tissues. In addition to colorimetric staining, fluorimetric staining that can be imaged by confocal microscopy can also be used with this protocol, thus providing full 3D resolution. The entire procedure can take <3 d from tissue preparation to image acquisition.
原位 mRNA 杂交是分析基因表达模式的最强大技术之一。然而，在需要固定、嵌入和切片样本以定位所需的 mRNA 之前，其在复杂的植物组织中的应用受到限制。在这里，我们提出了一种稳健的整体原位杂交方法，该方法允许在完整的复杂组织中轻松访问基因表达模式，例如拟南芥花序顶端。组织首先用经过优化的细胞壁消化酶混合物固定和渗透处理，该混合物既能保持组织结构的完整性，又能检测深层组织中的表达模式。除了比色染色外，还可以使用该方案进行可以通过共聚焦显微镜成像的荧光染色，从而提供全 3D 分辨率。从组织准备到图像采集，整个过程可以在<3 天内完成。
