Detection of calcitonin-encoding mRNA by radioactive and non- radioactive in situ hybridization: improved colorimetric detection and cellular localization of mRNA in thyroid sections

The localization of mRNA encoding calcitonin was studied by in situ hybridization using 35S-labeled RNA probes and biotin-labeled DNA probes. Radiolabeled probes were detected by autoradiography and biotin-labeled probes by streptavidin-biotin-peroxidase. To intensify the colorimetric signal, the in...

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Bibliographic Details
Published in:The journal of histochemistry and cytochemistry 1990-03, Vol.38 (3), p.351-358
Main Authors: Denijn, M, De Weger, RA, Berends, MJ, Compier-Spies, PI, Jansz, H, Van Unnik, JA, Lips, CJ
Format: Article
Language:English
Subjects:
Affinity Labels
Animals
Biological and medical sciences
Biotin - analysis
Calcitonin - analysis
Calcitonin - genetics
Colorimetry
Diverse techniques
Frozen Sections
Fundamental and applied biological sciences. Psychology
Genetic Code
Genetic Techniques
Immunohistochemistry
Molecular and cellular biology
Nucleic Acid Hybridization
Rats
RNA Probes - analysis
RNA, Messenger - analysis
Thyroid Gland - analysis
Thyroid Gland - ultrastructure
Tissue Preservation
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Summary:The localization of mRNA encoding calcitonin was studied by in situ hybridization using 35S-labeled RNA probes and biotin-labeled DNA probes. Radiolabeled probes were detected by autoradiography and biotin-labeled probes by streptavidin-biotin-peroxidase. To intensify the colorimetric signal, the indirect avidin-biotin complex (ABC) method was performed. However, the results were often variable. To improve the sensitivity, the peroxidase reaction signal was enhanced with a gold-silver deposit intensification reaction. To shorten the incubation times and to enhance the colorimetric reaction, several reaction steps were performed in a microwave oven. The localization of calcitonin mRNA in thyroid tissue, as detected with in situ hybridization, was confirmed by immunohistochemical localization of the calcitonin polypeptide. The results of in situ hybridization using biotinylated probes were compared to in situ hybridization using radioactive probes. Our data show that the results of in situ hybridization applied on frozen and paraffin-embedded sections using biotinylated DNA probes, detected with an indirect streptavidin-biotin-peroxidase reaction and intensified by silver-gold enhancement, were comparable to those obtained with radioactive probes. The localization of calcitonin encoding mRNA was in agreement with the localization of the calcitonin polypeptide.
ISSN:0022-1554
1551-5044
DOI:10.1177/38.3.2406337