Detection of two different mRNAs in a single section by dual in situ hybridization: a comparison between colorimetric and fluorescent detection.
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.
Barroso-Chinea
Pedro
P
Basal Ganglia Neuromorphology Laboratory, Neuroscience Division, Center for Applied Medical Research, University of Navarra Medical College, Pio XII Avenue 55, 31008 Pamplona, Spain.
Aymerich
María S
MS
Castle
María M
MM
Pérez-Manso
Mónica
M
Tuñón
Teresa
T
Erro
Elena
E
Lanciego
José L
JL
D000818:Animals; D001710:Biotin; D001921:Brain / Q000166:cytology / Q000502:physiology*; D003124:Colorimetry / Q000379:methods; D005784:Gene Amplification; D051273:Glucose Transporter Type 2 / Q000235:genetics; D005968:Glutamate Decarboxylase / Q000235:genetics; D017403:In Situ Hybridization / Q000379:methods; D007202:Indicators and Reagents; D007527:Isoenzymes / Q000235:genetics; D008297:Male; D010957:Plasmids; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D013050:Spectrometry, Fluorescence / Q000379:methods
D051273:Glucose Transporter Type 2; D007202:Indicators and Reagents; D007527:Isoenzymes; D012333:RNA, Messenger; C495683:Slc2a2 protein, rat; D001710:Biotin; D005968:Glutamate Decarboxylase; C402565:glutamate decarboxylase 1