The visualization of multiple gene expressions in well-preserved tissues is crucial for the elucidation of physiological and pathological processes. In situ hybridization chain reaction (HCR) is a method to visualize specific mRNAs in diverse organisms by applying a HCR that is an isothermal enzyme-free nucleotide polymerization method using hairpin DNAs. Although in situ HCR is a versatile method, this method is not widely used by researchers because of their higher cost than conventional in situ hybridization (ISH). Here, we redesigned hairpin DNAs so that their lengths were half the length of commonly used hairpin DNAs. We also optimized the conjugated fluorophores and linkers. Modified in situ HCR showed sufficient fluorescent signals to detect various mRNAs such as Penk, Oxtr, Vglut2, Drd1, Drd2, and Moxd1 in mouse neural tissues with a high signal-to-noise ratio. The sensitivity of modified in situ HCR in detecting the Oxtr mRNA was better than that of fluorescent ISH using tyramide signal amplification. Notably, the modified in situ HCR does not require proteinase K treatment so that it enables the preservation of morphological structures and antigenicity. The modified in situ HCR simultaneously detected the distributions of c-Fos immunoreactivity and Vglut2 mRNA, and detected multiple mRNAs with a high signal-noise ratio at subcellular resolution in mouse brains. These results suggest that the modified in situ HCR using short hairpin DNAs is cost-effective and useful for the visualization of multiple mRNAs and proteins.

保存完好的组织中多个基因表达的可视化对于阐明生理和病理过程至关重要。原位杂交链式反应 (HCR) 是一种通过应用 HCR 来可视化不同生物体中特定 mRNA 的方法，HCR 是一种使用发夹 DNA 的等温无酶核苷酸聚合方法。虽然就地HCR是一种通用方法，但由于其成本比传统方法高，因此并未被研究人员广泛使用就地杂交（ISH）。在这里，我们重新设计了发夹 DNA，使其长度是常用发夹 DNA 长度的一半。我们还优化了共轭荧光团和接头。修改的就地HCR 显示出足够的荧光信号来检测各种 mRNA，例如彭克,奥克斯特尔,谷胱甘肽2 ,博士1 ,博士2 ， 和莫克斯德1在小鼠神经组织中具有高信噪比。修改后的灵敏度就地HCR 检测奥克斯特尔mRNA 优于使用酪酰胺信号放大的荧光 ISH。值得注意的是，修改后的就地HCR 不需要蛋白酶 K 处理，因此能够保留形态结构和抗原性。修改后的就地HCR同时检测c-Fos免疫反应性的分布和谷胱甘肽2 mRNA，并在小鼠大脑中以亚细胞分辨率检测到多个具有高信噪比的 mRNA。 这些结果表明修改后的就地使用短发夹 DNA 的 HCR 具有成本效益，并且对于多种 mRNA 和蛋白质的可视化非常有用。