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Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry

Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ... In the present study some experimental parameters for in situ hybridization histochemistry (ISHH) have been analysed using35S-labelled and alkaline phosphatase-conjugated probes, in order to develop a reproducible double-labelling procedure. We have compared the total exclusion of tissue fixation with tissue sections fixed by immersion in formalin. In addition, the effect of dithiothreitol was assessed both when combining radiolabelled and non-radioactive probes on a single tissue section and when the probes were used separately. Hybridization of unfixed tissue resulted in stronger specific labelling and lower background both for radiolabelled and alkaline phosphatase-conjugated probes. No loss in tissue preservation was seen at the light microscopic level after hybridization of unfixed tissue. High concentrations (200 mM) of dithiothreitol strongly suppressed background when using35S-labelled probes, whereas in the non-radioactive procedure, alkaline phosphatase labelling could only be achieved with very low dithiothreitol concentrations (<1 mM). This incompatibility led to a protocol using unfixed tissue sections and a sequential hybridization procedure, with the radiolabelled probe and high concentrations of dithiothreitol in the first step and the alkaline phosphatase-conjugated probe without dithiothreitol in the second step. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Histochemistry and Cell Biology Springer Journals

Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry

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References (58)

Publisher
Springer Journals
Copyright
Copyright © 1992 by Springer-Verlag
Subject
Biomedicine; Biomedicine, general; Cell Biology; Biochemistry, general; Developmental Biology
ISSN
0948-6143
eISSN
1432-119X
DOI
10.1007/BF00716936
Publisher site
See Article on Publisher Site

Abstract

In the present study some experimental parameters for in situ hybridization histochemistry (ISHH) have been analysed using35S-labelled and alkaline phosphatase-conjugated probes, in order to develop a reproducible double-labelling procedure. We have compared the total exclusion of tissue fixation with tissue sections fixed by immersion in formalin. In addition, the effect of dithiothreitol was assessed both when combining radiolabelled and non-radioactive probes on a single tissue section and when the probes were used separately. Hybridization of unfixed tissue resulted in stronger specific labelling and lower background both for radiolabelled and alkaline phosphatase-conjugated probes. No loss in tissue preservation was seen at the light microscopic level after hybridization of unfixed tissue. High concentrations (200 mM) of dithiothreitol strongly suppressed background when using35S-labelled probes, whereas in the non-radioactive procedure, alkaline phosphatase labelling could only be achieved with very low dithiothreitol concentrations (<1 mM). This incompatibility led to a protocol using unfixed tissue sections and a sequential hybridization procedure, with the radiolabelled probe and high concentrations of dithiothreitol in the first step and the alkaline phosphatase-conjugated probe without dithiothreitol in the second step.

Journal

Histochemistry and Cell BiologySpringer Journals

Published: Nov 17, 2004

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