Komatsu Yoshihiro, Kishigami Satoshi, Mishina Yuji
School of Biology-Oriented Science and Technology, Kinki University, Kinki, Japan.
Methods Mol Biol. 2014;1092:1-15. doi: 10.1007/978-1-60327-292-6_1.
In situ hybridization is a powerful method for detecting endogenous mRNA sequences in morphologically preserved samples. We provide in situ hybridization methods, which are specifically optimized for mouse embryonic samples as whole mounts and section tissues. Additionally, β-Galactosidase (β-gal) is a popular reporter for detecting the expression of endogenous or exogenous genes. We reveal that 6-chloro-3-indoxyl-β-D-galactopyranoside (S-gal) is a more sensitive substrate for β-gal activity than 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal). S-gal is advantageous where β-gal activity is limited including early stage mouse embryos. As a result of the increased sensitivity as well as the color compatibility of S-gal, we successfully combined β-gal staining using S-gal with in situ hybridization using DIG-labeled probes in both whole mounts and sections.
原位杂交是一种用于在形态学保存完好的样本中检测内源性mRNA序列的强大方法。我们提供了原位杂交方法，这些方法是专门针对小鼠胚胎样本作为整体标本和切片组织进行优化的。此外，β-半乳糖苷酶（β-gal）是一种用于检测内源性或外源性基因表达的常用报告基因。我们发现，6-氯-3-吲哚基-β-D-吡喃半乳糖苷（S-gal）比5-溴-4-氯-3-吲哚基-β-D-半乳糖苷（X-gal）是一种对β-gal活性更敏感的底物。在β-gal活性有限的情况下，包括早期小鼠胚胎，S-gal具有优势。由于S-gal的灵敏度提高以及颜色兼容性，我们成功地将使用S-gal的β-gal染色与使用地高辛标记探针的原位杂交在整体标本和切片中结合起来。
