原位杂交
小RNA
生物
原位
荧光原位杂交
计算生物学
免疫荧光
细胞生物学
蛋白酶
分子生物学
基因表达
生物化学
遗传学
化学
基因
酶
抗体
有机化学
染色体
作者
Jente R. A. Boen,Isabel Pintelon,Andreas B. Gevaert,Vincent F. M. Segers,Emeline M. Van Craenenbroeck
摘要
Abstract The last decades have illustrated the importance of microRNAs (miRNAs) in various biological and pathological processes. The combined visualization of miRNAs using fluorescent in situ hybridization (FISH) and proteins using immunofluorescence (IF) can reveal their spatiotemporal distribution in relation to the cell and tissue morphology and can provide interesting insights into miRNA‐protein interactions. However, standardized protocols for co‐localization of miRNAs and proteins are currently lacking, and substantial technical obstacles still need to be addressed. In particular, the incompatibility of protein IF protocols with steps required for miRNA FISH, such as proteolytic pretreatments and ethylcarbodiimide post‐fixation, as well as hurdles related to low signal intensity of low‐copy miRNAs, remains challenging. Our technique may considerably enhance miRNA‐based research, as current detection techniques lack the ability to elucidate cellular and subcellular localization. Here, we describe an optimized 2‐day protocol for combined detection of low‐abundant miRNAs and proteins in cryosections of cardiac tissue, without the need for protease‐dependent pretreatment or post‐fixation treatment. We successfully demonstrate endothelial‐specific localization of low‐abundant miR‐181c‐5p in cardiac tissue. © 2023 Wiley Periodicals LLC. Basic Protocol : Fluorescent in situ hybridization for miRNA combined with staining of proteins
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