A supersandwich fluorescence in situ hybridization strategy for highly sensitive and selective mRNA imaging in tumor cells

2016 ◽  
Vol 52 (2) ◽  
pp. 370-373 ◽  
Author(s):  
Jin Huang ◽  
He Wang ◽  
Xiaohai Yang ◽  
Yanjing Yang ◽  
Ke Quan ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Tumor Cells ◽  
Signal Amplification ◽  
Fluorescence Signal ◽  
Target Mrna ◽  
Highly Sensitive ◽  
Fluorescence Signal Amplification

This strategy uses two fluorophore-labeled signal probes to generate a supersandwich product, which in turn generates numerous signal probes located at the target mRNA position, resulting in thein situfluorescence signal amplification.

Imaging of intracellular-specific microRNA in tumor cells by symmetric exponential amplification-assisted fluorescence in situ hybridization

2018 ◽  
Vol 54 (99) ◽  
pp. 13981-13984 ◽  
Author(s):  
Jun Chen ◽  
Wen Yin ◽  
Yingjun Ma ◽  
Huihui Yang ◽  
Yanfei Zhang ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Tumor Cells

A symmetric exponential amplification-assisted fluorescence in situ hybridization (SEXPAR-FISH) strategy was reported for imaging intracellular-specific microRNAs.

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.

scholarly journals Catalyzed Reported Deposition-Fluorescence In Situ Hybridization Protocol To Evaluate Phagotrophy in Mixotrophic Protists

2005 ◽  
Vol 71 (11) ◽  
pp. 7321-7326 ◽  
Author(s):  
Juan M. Medina-Sánchez ◽  
Marisol Felip ◽  
Emilio O. Casamayor
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Signal Amplification ◽  
Cell Attachment ◽  
Cell Loss ◽  
Alexa Fluor ◽  
Hybridization Protocol ◽  
Card Fish ◽  
First Time

ABSTRACT We describe a catalyzed reported deposition-fluorescence in situ hybridization (CARD-FISH) protocol particularly suited to assess the phagotrophy of mixotrophic protists on prokaryotes, since it maintains cell and plastid integrity, avoids cell loss and egestion of prey, and allows visualization of labeled prey against plastid autofluorescence. This protocol, which includes steps such as Lugol's-formaldehyde-thiosulfate fixation, agarose cell attachment, cell wall permeabilization with lysozyme plus achromopeptidase, and signal amplification with Alexa-Fluor 488, allowed us to detect almost 100% of planktonic prokaryotes (Bacteria and Archaea) and, for the first time, to show archaeal cells ingested by mixotrophic protists.

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