Sensitive Whole-Mount Fluorescent In Situ Hybridization in Zebrafish Using Enhanced Tyramide Signal Amplification

2013 ◽  
pp. 175-185 ◽  
Author(s):  
Gilbert Lauter ◽  
Iris Söll ◽  
Giselbert Hauptmann
Keyword(s):  
In Situ Hybridization ◽  
Signal Amplification ◽  
Fluorescent In Situ Hybridization ◽  
Tyramide Signal Amplification ◽  
Whole Mount

Horse Radish Peroxidase Labelled Oligonucleotides and Tyramide Signal Amplification for Fluorescence in Situ Hybridization to Low Abundant Cytokine Mrnas

1997 ◽  
Vol 3 (S2) ◽  
pp. 203-204
Author(s):  
Mariette van de Corput ◽  
Rob van Gijlswijk ◽  
Mark Bobrow ◽  
Tom Erickson ◽  
Roel Dirks ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Signal Amplification ◽  
Signal To Noise Ratio ◽  
High Specificity ◽  
Tyramide Signal Amplification ◽  
Horse Radish Peroxidase ◽  
Metaphase Chromosomes ◽  
Generation Capacity

In recent years, Tyramide Signal Amplification (TSA) has gained acclaim as a very sensitive detection method for immunocytochemsitry and fluorescence in situ hybridization (FISH). To maximally exploit the great signal generation capacity of TSA in mRNA-FISH, minimizing signals emanating from non-specifically bound nucleic acid probe becomes of prime importance, because a specificity check of the signals observed in the cytoplasm is virtually impossible. We reasoned that utilization of synthetic oligonucleotides (ONTs) in stead of commonly used cDNAs or cRNAs would diminish non-specific probe binding and that direct Horse Radish Peroxidase (HRP) labelling of ONTs and TSA would enable their in situ detection.This approach was first tested in metaphase DNA-FISH using chromosome-specific repeats as targets. Using bifunctional crosslinking chemistry and HPLC, 5’-hexylamino oligonucleotides for chromosome specific simple satellite and alphoid sequences were conjugated to HRP and purified. Following 15 - 20 min of situ hybridization of a single HRP-ONT probe to metaphase chromosomes and a direct flurochrome-tyramide detection step, such repeat targets could be visualized with high specificity and excellent signal-to noise ratio.

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