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Double and triple in situ hybridization for coexpression studies: combined fluorescent and chromogenic detection of neuropeptide Y (NPY) and serotonin receptor subtype mRNAs expressed at different abundance levels

Please always quote using this URN: urn:nbn:de:bvb:20-opus-126720
  • Multiple fluorescence in situ hybridization is the method of choice for studies aimed at determining simultaneous production of signal transduction molecules and neuromodulators in neurons. In our analyses of the monoamine receptor mRNA expression of peptidergic neurons in the rat telencephalon, double tyramide-signal-amplified fluorescence in situ hybridization delivered satisfactory results for coexpression analysis of neuropeptide Y (NPY) and serotonin receptor 2C (5-HT2C) mRNA, a receptor subtype expressed at high-to-moderate abundance inMultiple fluorescence in situ hybridization is the method of choice for studies aimed at determining simultaneous production of signal transduction molecules and neuromodulators in neurons. In our analyses of the monoamine receptor mRNA expression of peptidergic neurons in the rat telencephalon, double tyramide-signal-amplified fluorescence in situ hybridization delivered satisfactory results for coexpression analysis of neuropeptide Y (NPY) and serotonin receptor 2C (5-HT2C) mRNA, a receptor subtype expressed at high-to-moderate abundance in the regions analyzed. However, expression of 5-HT1A mRNA, which is expressed at comparatively low abundance in many telencephalic areas, could not be unequivocally identified in NPY mRNA-reactive neurons due to high background and poor signal-to-noise ratio in fluorescent receptor mRNA detections. Parallel chromogenic in situ hybridization provided clear labeling for 5-HT1A mRNA and additionally offered the possibility to monitor the chromogen deposition at regular time intervals to determine the optimal signal-to-noise ratio. We first developed a double labeling protocol combining fluorescence and chromogenic in situ hybridization and subsequently expanded this variation to combine double fluorescence and chromogenic in situ hybridization for triple labelings. With this method, we documented expression of 5-HT2C and/or 5-HT1A in subpopulations of telencephalic NPY-producing neurons. The method developed in the present study appears suitable for conventional light and fluorescence microscopy, combines advantages of fluorescence and chromogenic in situ hybridization protocols and thus provides a reliable non-radioactive alternative to previously published multiple labeling methods for coexpression analyses in which one mRNA species requires highly sensitive detection.show moreshow less

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Metadaten
Author: Maria Bonn, Angelika Schmitt, Esther Asan
URN:urn:nbn:de:bvb:20-opus-126720
Document Type:Journal article
Faculties:Medizinische Fakultät / Institut für Anatomie und Zellbiologie
Medizinische Fakultät / Klinik und Poliklinik für Psychiatrie, Psychosomatik und Psychotherapie
Language:English
Parent Title (English):Histochemistry and Cell Biology
Year of Completion:2012
Volume:137
Issue:1
Pagenumber:11-24
Source:Histochemistry and Cell Biology (2012) 137:11–24 DOI 10.1007/s00418-011-0882-3
DOI:https://doi.org/10.1007/s00418-011-0882-3
Dewey Decimal Classification:6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit
Tag:5-HT1A; 5-HT2C; Coexpression; NPY; triple in situ hybridization
Release Date:2016/02/09
Licence (German):License LogoCC BY-NC: Creative-Commons-Lizenz: Namensnennung, Nicht kommerziell