PUBLICATION
Sensitive Multiplexed Fluorescent In Situ Hybridization Using Enhanced Tyramide Signal Amplification and Its Combination with Immunofluorescent Protein Visualization in Zebrafish
- Authors
- Lauter, G., Söll, I., Hauptmann, G.
- ID
- ZDB-PUB-190926-5
- Date
- 2020
- Source
- Methods in molecular biology (Clifton, N.J.) 2047: 397-409 (Review)
- Registered Authors
- Hauptmann, Giselbert, Söll, Iris
- Keywords
- FISH, Fluorescent in situ hybridization, Immunofluorescence, Peroxidase, RNA, TSA, Transcript, Tyramide signal amplification, Zebrafish
- MeSH Terms
-
- Alkaline Phosphatase/metabolism
- Animals
- Embryo, Nonmammalian/metabolism
- Horseradish Peroxidase/metabolism
- Immunoassay
- In Situ Hybridization, Fluorescence/methods*
- RNA, Messenger/metabolism
- Zebrafish/embryology
- Zebrafish/metabolism*
- PubMed
- 31552667 Full text @ Meth. Mol. Biol.
Citation
Lauter, G., Söll, I., Hauptmann, G. (2020) Sensitive Multiplexed Fluorescent In Situ Hybridization Using Enhanced Tyramide Signal Amplification and Its Combination with Immunofluorescent Protein Visualization in Zebrafish. Methods in molecular biology (Clifton, N.J.). 2047:397-409.
Abstract
Fluorescent in situ hybridization (FISH) provides sensitive detection and visualization of RNA transcripts in tissues and cells with high resolution. We present here a multiplex RNA FISH method using enhanced tyramide signal amplification (TSA) for colocalization analysis of three different transcripts in intact zebrafish brains. To achieve enhancement of fluorescent signals, essential steps of the FISH procedure are optimized including embryo permeability, hybridization efficacy, and fluorogenic TSA-reaction conditions. Critical to this protocol, the enzymatic peroxidase (PO) reactivity is significantly improved by the application of viscosity-increasing polymers, PO accelerators, and highly effective bench-made tyramide substrates. These advancements lead to an optimized TSA-FISH protocol with dramatically increased signal intensity and signal-to-background ratio allowing for visualization of three mRNA transcript patterns simultaneously. The TSA-FISH procedure can be combined with immunofluorescence (IF) to compare mRNA transcript and protein expression patterns.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping