Hostname: page-component-cd9895bd7-jkksz Total loading time: 0 Render date: 2024-12-27T11:21:48.188Z Has data issue: false hasContentIssue false
  • English
  • Français

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

Published online by Cambridge University Press:  02 July 2020

Mariette van de Corput ,
Rob van Gijlswijk ,
Mark Bobrow ,
Tom Erickson ,
Roel Dirks  and
Ton Raap
Mariette van de Corput
Affiliation:
Laboratory for Cytochemistry and Cytometry, Leiden University Medical Center, Leiden, The Netherlands
Rob van Gijlswijk
Affiliation:
Laboratory for Cytochemistry and Cytometry, Leiden University Medical Center, Leiden, The Netherlands
Mark Bobrow
Affiliation:
NEN-Life Science Products, Boston, USA
Tom Erickson
Affiliation:
NEN-Life Science Products, Boston, USA
Roel Dirks
Affiliation:
Laboratory for Cytochemistry and Cytometry, Leiden University Medical Center, Leiden, The Netherlands
Ton Raap
Affiliation:
Laboratory for Cytochemistry and Cytometry, Leiden University Medical Center, Leiden, The Netherlands
Get access

Extract

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.

Type
Genomes, Chromsomes and Genes: Emerging Technologies
Information
Microscopy and Microanalysis , Volume 3 , Issue S2: Proceedings: Microscopy & Microanalysis '97, Microscopy Society of America 55th Annual Meeting, Microbeam Analysis Society 31st Annual Meeting, Histochemical Society 48th Annual Meeting, Cleveland, Ohio, August 10-14, 1997 , August 1997 , pp. 203 - 204
Copyright
Copyright © Microscopy Society of America 1997

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

1.Adams, J.C., Journal of Histochemistry and Cytochemistry (1992) 40, 1457.10.1177/40.10.1527370CrossRefGoogle Scholar
2.Berghorn, K.A. et al, Journal of Histochemistry and Cytochemistry (1994) 42, 1635–1642.10.1177/42.12.7983364CrossRefGoogle Scholar
3.Bobrow, M.N. et al, Journal of Immunological Methods (1991) 137, 103–112.10.1016/0022-1759(91)90399-ZCrossRefGoogle Scholar
4.Bobrow, M.N. et al, Journal of Immunological Methods (1992) 150, 145–149.10.1016/0022-1759(92)90073-3CrossRefGoogle Scholar
5.Bobrow, M.N. et al, Journal of Immunological Methods (1992) 137, 103–112.10.1016/0022-1759(91)90399-ZCrossRefGoogle Scholar
6.Dirks, R.W. et al, Journal of Cell Science (1993) 104, 1187–1197.Google Scholar
7.Gijlswijk, R.P.M. van et al, Journal of Histochemistry and Cytochemistry (1996) 44, 389–392.10.1177/44.4.8601698CrossRefGoogle Scholar
8.Gijlswijk, R.P.M. Van et al, Cytogenetics Cell Genetics (1996) 75, 258–262.10.1159/000134496CrossRefGoogle Scholar
9.Hunyady, B., Journal of Histochemistry and Cytochemistry (1996) 4, 1353–1362.10.1177/44.12.8985127CrossRefGoogle Scholar
10.Kaashoek, J. et al, Lymphokine and Cytokine Rtesearch (1991) 10, 231–235Google Scholar
11.Kerstens, H.M. et al, Journal of Histochemistry and Cytochemistry(1995) 43, 347–352.10.1177/43.4.7897179CrossRefGoogle Scholar
12.Raap, A.K. et al, Human Molecular Genetics (1995) 4, 529–534.10.1093/hmg/4.4.529CrossRefGoogle Scholar
13.Shindler, K.S. and Roth, K., Journal of Histochemistry and Cytochemistry (1996) 44, 1331–1335.10.1177/44.11.8918908CrossRefGoogle Scholar
14.This work was supported in part by the Dutch Science Organization NWO (area Medical Sciences, Project 900-534-109) and NEN-Life Science Products.Google Scholar