Detection and signal amplification in zebrafish RNA FISH

Methods : a Companion to Methods in Enzymology
Giselbert HauptmannIris Söll

Abstract

In situ hybridization (ISH) has become an invaluable tool for the detection of RNA in cells, tissues and organisms. Due to improvements in target and signal amplification and in probe design remarkable progress has been made concerning sensitivity, specificity and resolution of chromogenic and fluorescent ISH (FISH). These advancements allow for exquisite cellular and sub-cellular resolution and for detecting multiple RNA species at a time by multiplexing. In zebrafish (F)ISH non-enzymatic and enzymatic amplification systems have been employed to obtain enhanced signal intensities and signal-to-noise ratios. These amplification strategies include branched DNA-based RNAscope and in situ hybridization chain reaction (HCR) techniques, as well as alkaline phosphatase (AP)- and horseradish peroxidase (PO)-based immunoassays. For practical application, we provide proven multiplex FISH protocols for AP- and PO-based visualization of mRNAs at high resolution. The protocols take advantage of optimized tyramide signal amplification (TSA) conditions of the PO assay and long-lasting high signal-to-noise ratio of the AP reaction, thereby enabling detection of less abundant transcripts.

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Citations

Mar 2, 2016·Methods : a Companion to Methods in Enzymology·Yaron Shav-Tal
Sep 23, 2016·Frontiers in Cell and Developmental Biology·Chenghua CuiPeining Li
Oct 27, 2016·International Journal of Obesity : Journal of the International Association for the Study of Obesity·J ChenX Wang
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May 19, 2017·Applied Biochemistry and Biotechnology·Tahereh FozooniHosseinali Sasan
Dec 16, 2016·Nature Reviews. Neuroscience·Birte U ForstmannMax C Keuken
Jun 13, 2021·Journal of Ovarian Research·Liping WangYuanhang Zhu

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