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PMID: 1429016 Published · ppublish English Comparative Study Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

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

Histochemistry ·Vol. 98 ·No. 1 ·1992-08-00 ·Pages 39-49

Dagerlind A, Friberg K, Bean AJ, Hökfelt T

Abstract

In the present study some experimental parameters for in situ hybridization histochemistry (ISHH) have been analysed using 35S-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 using 35S-labelled probes, whereas in the non-radioactive procedure, alkaline phosphatase labelling could only be achieved with very low dithiothreitol concentrations (less than 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.

MeSH Terms
Alkaline Phosphatase Animals Autoradiography Formaldehyde Histocytochemistry In Situ Hybridization Male Microscopy/methods RNA Probes RNA, Messenger/analysis Rats Rats, Sprague-Dawley Sensitivity and Specificity Sulfur Radioisotopes Tissue Fixation
Chemicals
RNA Probes RNA, Messenger Sulfur Radioisotopes Formaldehyde Alkaline Phosphatase
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Dagerlind A
Department of Histology and Neurobiology, Karolinska Institute, Stockholm, Sweden.
Friberg K
Bean A J
Hökfelt T
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Article Info
Journal
Histochemistry
Abbr.
Histochemistry
ISSN
0301-5564
DOI
10.1007/BF00716936
Published
1992-08-00
Pages
39-49
Language
English
Region
Germany
NLM ID
0411300
Subset
IM
Grants
NIMH NIH HHS · MH 43230-02 · United States
External Links
PubMed source DOI: 10.1007/BF00716936
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