Department of Medicine, Division of Genetic Medicine, Vanderbilt University Medical Center, Nashville, Tennessee.
Curr Protoc. 2022 May;2(5):e439. doi: 10.1002/cpz1.439.
In situ hybridization has been a robust method for detection of mRNA expression in whole-mount samples or tissue sections for more than 50 years. Recent technical advances for in situ hybridization have incorporated oligo-based probes that attain greater tissue penetration and signal amplification steps with restricted localization for visualization of specific mRNAs within single cells. One such method is third-generation in situ hybridization chain reaction (V3HCR). Here, we report an optimized protocol for V3HCR detection of gene expression using sectioned frozen tissues from mouse and human on microscope slides. Our methods and modifications for cryosectioning, tissue fixation, and processing over a three-day V3HCR protocol are detailed along with recommendations for aliquoting and storing V3HCR single-stranded DNA probes and hairpin amplifiers. In addition, we describe a method for blocking background signal from lipofuscin, a highly autofluorescent material that is widespread in human neurons and often complicates imaging efforts. After testing multiple strategies for reduction of lipofuscin, we determined that application of a lipofuscin quencher dye is compatible with V3HCR, in contrast to other methods like cupric sulfate quenching or Sudan Black B blocking that cause V3HCR signal loss. This adaptation enables application of V3HCR for in situ detection of gene expression in human neuronal populations that are otherwise problematic due to lipofuscin autofluorescence. © 2022 Wiley Periodicals LLC. Basic Protocol: Mouse and human fresh-frozen tissue in situ hybridization chain reaction on microscope slides Support Protocol: Aliquoting of HCR probes and hairpins.
原位杂交技术已经成为一种强大的方法，可用于检测 50 多年来全组织样本或组织切片中的 mRNA 表达。最近，原位杂交技术的技术进步已经将寡核苷酸探针整合在一起，这些探针具有更大的组织穿透性和信号放大步骤，并通过限制特定 mRNA 在单个细胞内的定位来实现可视化。其中一种方法是第三代原位杂交链反应（V3HCR）。在这里，我们报告了一种优化的 V3HCR 检测方法，用于在显微镜载玻片上检测来自小鼠和人类的冷冻组织切片中的基因表达。我们详细介绍了用于冷冻切片、组织固定和为期三天的 V3HCR 方案处理的方法和改进，以及 V3HCR 单链 DNA 探针和发夹扩增剂的分装和储存建议。此外，我们还描述了一种阻断脂褐素背景信号的方法，脂褐素是一种高度自发荧光的物质，广泛存在于人类神经元中，经常使成像工作复杂化。在测试了多种减少脂褐素的策略后，我们确定脂褐素淬灭染料的应用与 V3HCR 兼容，而其他方法，如硫酸铜淬灭或苏丹黑 B 阻断，会导致 V3HCR 信号丢失。这种适应能力使 V3HCR 能够用于检测人类神经元群体中的基因表达，否则由于脂褐素的自发荧光，这些群体的检测会很困难。© 2022 威利父子公司。基本方案：显微镜载玻片上的小鼠和人类新鲜冷冻组织原位杂交链反应支持方案：HCR 探针和发夹的分装。
