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Dadun > Depósito Académico > CIMA (Centro de Investigación Médica Aplicada) > Área de Neurociencias > Neuroanatomía ganglios basales > DA - CIMA - Neurociencias - Neuroanatomía ganglios basales - Artículos de Revista >

Detection of two different mRNAs in a single section by dual in situ hybridization: a comparison between colorimetric and fluorescent detection
Authors: Barroso-Chinea, P. (P.)
Aymerich, M.S. (María S.)
Castle, M. (M.)
Perez-Manso, M. (Mónica)
Tuñon, T. (Teresa)
Lanciego, J. (J.L.)
Keywords: Digoxigenin
Biotin
Riboprobe
mRNA
VGLUT;
GAD67
GAD65
Issue Date: 2007
Publisher: Elsevier
Publisher version: http://www.sciencedirect.com/science/article/pii/S0165027007000040
ISSN: 1872-678X
Citation: Barroso-Chinea P, Aymerich MS, Castle MM, Perez-Manso M, Tuñon T, Erro E, et al. Detection of two different mRNAs in a single section by dual in situ hybridization: a comparison between colorimetric and fluorescent detection. J Neurosci Methods 2007 May 15;162(1-2):119-128.
Abstract
We have compared the performance of two methods designed to simultaneously detect two different mRNAs within a single brain section by dual ISH. Specific mRNA riboprobes labeled with biotin and digoxigenin were simultaneously hybridized and visualized using either brightfield or fluorescence microscopy. For brightfield visualization, the biotin-labeled riboprobe was detected with a peroxidase chromogen, whereas, an alkaline phosphatase substrate was used for the detection of the digoxigenin-labeled riboprobe. Dual fluorescent ISH involved the detection of the biotin-labeled riboprobe with an Alexa((R))488-conjugated streptavidin followed by the visualization of the digoxigenin-labeled riboprobe with the red fluorescent substrate HNPP. The dual ISH protocols presented here offer sensitive methods to detect the expression of two mRNAs of interest, with both colorimetric and fluorescent ISH each having its strengths and limitations. For example, dual colorimetric ISH has proven to be particularly useful to study the distribution of two mRNAs in different brain nuclei, whereas, dual fluorescent ISH has provided better results when studying the co-localization of two different mRNAs in single neurons. The comprehensive step-by-step procedure is presented, together with a troubleshooting section in which the advantages and limitations of these procedures are reviewed in depth. Moreover, alternative protocols for dual ISH were also compared to those presented here.
Permanent link: http://hdl.handle.net/10171/20097
Appears in Collections:DA - Ciencias - HAP - Artículos de revista
DA - CIMA - Neurociencias - Neuroanatomía ganglios basales - Artículos de Revista

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