Work in the authors' laboratories was supported by the UK Biotechnology and Biological Sciences Research Council (BB/J006602/1 and BB/L003236/1), the Wellcome Trust (WT100269MA) and the European Research Council (ERC, "Ideas" Programme 293544). SJ was supported by an EMBO long-term fellowship. The authors thank Stephen Grant for his technical assistance.

Ethics Statement: 
Mouse husbandry and handling are in accordance with UK Home Office regulations and UCL ethics committee guidelines, complying with the Animals (Scientific Procedures) Act 1986 of the United Kingdom and its Amendment Regulations 2012.
NOTE: All solutions should be made with diethyl pyrocarbonate (DEPC)- treated water to destroy any residual RNase. For DEPC treatment, add DEPC (1 ml per litre), shake vigorously until all the DEPC globules have disappeared then autoclave t...

<ol>
	<li>Lobsiger, C. S., Cleveland, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+cells+as+intrinsic+components+of+non-cell-autonomous+neurodegenerative+disease.">Glial cells as intrinsic components of non-cell-autonomous neurodegenerative disease.</a> Nat Neurosci. 10 (11), 1355-1360 (2007).</li><li>Baslow, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-acetylaspartate+in+the+vertebrate+brain:+metabolism+and+function.">N-acetylaspartate in the vertebrate brain: metabolism and function.</a> Neurochem Res. 28 (6), 941-953 (2003).</li><li>Hoshino, H., Kubota, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Canavan+disease:+clinical+features+and+recent+advances+in+research.">Canavan disease: clinical features and recent advances in research.</a> Pediatr Int. 56 (4), 477-483 (2014).</li><li>Kaul, R., Gao, G. P., Balamurugan, K., Matalon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+of+the+human+aspartoacylase+cDNA+and+a+common+missense+mutation+in+Canavan+disease.">Cloning of the human aspartoacylase cDNA and a common missense mutation in Canavan disease.</a> Nat Genet. 5 (2), 118-123 (1993).</li><li>Divry, P., Mathieu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspartoacylase+deficiency+and+N-acetylaspartic+aciduria+in+patients+with+Canavan+disease.">Aspartoacylase deficiency and N-acetylaspartic aciduria in patients with Canavan disease.</a> Am J Med Genet. 32 (4), 550-551 (1989).</li><li>Bartalini, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+diagnosis+of+Canavan+disease.">Biochemical diagnosis of Canavan disease.</a> Childs Nerv Syst. 8 (8), 468-470 (1992).</li><li>Moffett, J. R., Ross, B., Arun, P., Madhavarao, C. N., Namboodiri, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-Acetylaspartate+in+the+CNS:+from+neurodiagnostics+to+neurobiology.">N-Acetylaspartate in the CNS: from neurodiagnostics to neurobiology.</a> Prog Neurobiol. 81 (2), 89-131 (2007).</li><li>D'Adamo, A. F., Smith, J. C., Woiler, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+occurrence+of+N-acetylaspartate+amidohydrolase+(aminoacylase+II)+in+the+developing+rat.">The occurrence of N-acetylaspartate amidohydrolase (aminoacylase II) in the developing rat.</a> J Neurochem. 20 (4), 1275-1278 (1973).</li><li>Bhakoo, K. K., Craig, T. J., Styles, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+and+regional+distribution+of+aspartoacylase+in+rat+brain+tissue.">Developmental and regional distribution of aspartoacylase in rat brain tissue.</a> J Neurochem. 79 (1), 211-220 (2001).</li><li>Sommer, A., Sass, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+aspartoacylase+(ASPA)+and+Canavan.">Expression of aspartoacylase (ASPA) and Canavan.</a> Gene. 505 (2), 206-210 (2012).</li><li>Klugmann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+distribution+of+aspartoacylase+in+the+postnatal+rat+brain.">Identification and distribution of aspartoacylase in the postnatal rat brain.</a> Neuroreport. 14 (14), 1837-1840 (2003).</li><li>Madhavarao, C. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+localization+of+aspartoacylase+in+the+rat+central+nervous+system.">Immunohistochemical localization of aspartoacylase in the rat central nervous system.</a> J Comp Neurol. 472 (3), 318-329 (2004).</li><li>Hershfield, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspartoacylase+is+a+regulated+nuclear-cytoplasmic+enzyme.">Aspartoacylase is a regulated nuclear-cytoplasmic enzyme.</a> Faseb J. 20 (12), 2139-2141 (2006).</li><li>Moffett, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+aspartoacylase+expression+in+the+rat+central+nervous+system.">Extensive aspartoacylase expression in the rat central nervous system.</a> Glia. 59 (10), 1414-1434 (2011).</li><li>Kirmani, B. F., Jacobowitz, D. M., Kallarakal, A. T., Namboodiri, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspartoacylase+is+restricted+primarily+to+myelin+synthesizing+cells+in+the+CNS:+therapeutic+implications+for+Canavan+disease.">Aspartoacylase is restricted primarily to myelin synthesizing cells in the CNS: therapeutic implications for Canavan disease.</a> Brain Res Mol Brain Res. 107 (2), 176-182 (2002).</li><li>Kirmani, B. F., Jacobowitz, D. M., Namboodiri, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+increase+of+aspartoacylase+in+oligodendrocytes+parallels+CNS+myelination.">Developmental increase of aspartoacylase in oligodendrocytes parallels CNS myelination.</a> Brain Res Dev Brain Res. 140 (1), 105-115 (2003).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+RNA-sequencing+transcriptome+and+splicing+database+of+glia,+neurons,+and+vascular+cells+of+the+cerebral+cortex.">An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex.</a> J Neurosci. 34 (36), 11929-11947 (2014).</li></ol>

This protocol provides a step-by-step procedure for a double RNA in situ hybridization followed by immunostaining. We have used this protocol to confirm that Aspa is expressed in mature oligodendrocytes in several brain areas.
This multi-step procedure has many potential pitfalls that can affect sensitivity and should be avoided. First, all the solutions and storage buffers for the transcription reaction need to be RNase-free. Second, the choice of cDNA templates is important...

Glial cells, which are the most abundant cells in the central nervous system (CNS), comprise oligodendrocytes (the myelinating cells of CNS), oligodendrocytes precursors (OPs, also known as &#34;NG2 cells&#34;), astrocytes and microglia. There is growing interest in the functions of glial cells and their potential roles in neurological diseases1. For example, Canavan disease (CD) is a hereditary neurodegenerative disorder starting early in infancy with spongiform leukodystrophy and a progressive loss of neurons, leading to death usually before 10 years of age2,3. Mutations in the Aspartoacyclase (ASPA) gene that lead to drastically reduced ASPA activity4 in CD have been identified. ASPA is an enzyme catalysing the deacetylation of N-acetylaspartate (NAA), a molecule highly concentrated in the brain, generating acetate and aspartate 5-7. Many CD patients show higher levels of NAA due to lack of ASPA activity. Some studies speculate that NAA-derived acetate could be a major source of fatty acids/lipids in the brain during development and CD may result from decreased myelin synthesis during development caused by the failure of NAA to be broken down3,5,6.
ASPA is predominantly found in the kidney, liver and white matter of the brain, and given the important role of ASPA in CD, the cellular expression of this enzyme in the brain has been studied by several labs. By looking at ASPA enzymatic activity in the brain, earlier studies found that the increase in ASPA activity during brain development parallels the time course of myelination 8-10. At the cellular level, assays for enzymatic activity as well as in situ hybridization (ISH) and immunohistochemistry (IHC) analyses suggest that ASPA is mainly expressed in oligodendrocytes in the brain but not in neurons or astrocytes11-16. A few studies found that ASPA might also be expressed in microglia in the CNS12,14. So far data on ASPA expression in OPs are limited. According to a recent study where transcriptomes of different cell types in the mouse cerebral cortex including neurons, astrocytes, OPs, newly formed oligodendrocytes, myelinating oligodendrocytes, microglia, endothelial cells, and pericytes were analysed by RNA sequencing17, ASPA is exclusively expressed in oligodendrocytes, in particular in myelinating oligodendrocytes (http://web.stanford.edu/group/barres_lab/brain_rnaseq.html).&#160; Despite these studies on ASPA expression pattern in the brain, a number of uncertainties remain.
Different techniques can be used to study gene expression patterns. IHC is a commonly used method for detecting the functional product (i.e., protein) of a gene expression in tissue sections. Despite its great utility, this technique has limitations as its application and specificity are subject to the availability and specificity of the antibody needed. By comparison, ISH has the advantage of being able to reveal the expression of any gene at the mRNA level. However, it can be technically challenging to use several probes at the same time in order to localize a gene expression to specific cell types. In this article, we describe a protocol combining double fluorescence RNA in situ hybridization with fluorescence immunolabelling of a protein. We have used this set of techniques to examine the expression pattern of Aspa in mouse brain. This method allows the precise study of gene expression using confocal microscopy.

<table border="0" cellpadding="0" cellspacing="0" width="460">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:111px;">QIAprep&#174; Miniprep</td>
			<td style="width:111px;">Qiagen</td>
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			<td style="width:131px;"></td>
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			<td height="42" style="height:42px;width:111px;">Deionized formamide</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:111px;">F9037</td>
			<td style="width:131px;">for ISH blocking buffer</td>
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			<td height="42" style="height:42px;width:111px;">Sodium chloride</td>
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			<td height="21" style="height:21px;width:111px;">Trizma Base</td>
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			<td style="width:111px;">T1503</td>
			<td style="width:131px;"></td>
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			<td height="42" style="height:42px;width:111px;">Hydrochloric acid</td>
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		<tr height="84">
			<td height="84" style="height:84px;width:111px;">Sodium phosphate dibasic dihydrate</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:111px;">Yeast tRNA</td>
			<td style="width:111px;">Roche</td>
			<td style="width:111px;">10109495001</td>
			<td style="width:131px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:111px;">50x Denhardt&#39;s solution</td>
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			<td height="21" style="height:21px;width:111px;">Dextran sulfate</td>
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			<td height="42" style="height:42px;width:111px;">Aspa cDNA clone</td>
			<td style="width:111px;">Source Bioscience</td>
			<td style="width:111px;">IRAVp968C0654D</td>
			<td style="width:131px;"></td>
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			<td height="42" style="height:42px;width:111px;">SalI</td>
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			<td style="width:131px;"></td>
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			<td height="21" style="height:21px;width:111px;">Sodium acetate</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:111px;">S2889</td>
			<td style="width:131px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:111px;">Equilibrated phenol</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:111px;">P4557</td>
			<td style="width:131px;"></td>
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			<td height="21" style="height:21px;width:111px;">Chloroform</td>
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			<td height="21" style="height:21px;width:111px;">Isoamyl alcohol</td>
			<td style="width:111px;">Aldrich</td>
			<td style="width:111px;">496200</td>
			<td style="width:131px;"></td>
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			<td height="42" style="height:42px;width:111px;">Ethanol</td>
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			<td style="width:111px;">20821.321</td>
			<td style="width:131px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:111px;">T7 RNA polymerase</td>
			<td style="width:111px;">Promega</td>
			<td style="width:111px;">P4074</td>
			<td style="width:131px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:111px;">Transcription buffer</td>
			<td style="width:111px;">Promega</td>
			<td style="width:111px;">P118B</td>
			<td style="width:131px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:111px;">100mM DTT</td>
			<td style="width:111px;">Promega</td>
			<td style="width:111px;">P117B</td>
			<td style="width:131px;"></td>
		</tr>
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			<td height="42" style="height:42px;width:111px;">UTP-DIG NTP mix</td>
			<td style="width:111px;">Roche</td>
			<td style="width:111px;">11277073910</td>
			<td style="width:131px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:111px;">Rnasin</td>
			<td style="width:111px;">Promega</td>
			<td style="width:111px;">N251B</td>
			<td style="width:131px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:111px;">Paraformaldehyde</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:111px;">P6148</td>
			<td style="width:131px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:111px;">Filter paper</td>
			<td style="width:111px;">Fisher scientific</td>
			<td style="width:111px;">005479470</td>
			<td style="width:131px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:111px;">Sucrose</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:111px;">59378</td>
			<td style="width:131px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:111px;">Diethyl pyrocarbonate</td>
			<td style="width:111px;">Sigma</td>
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			<td style="width:131px;"></td>
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			<td height="21" style="height:21px;width:111px;">Pentobarbitone</td>
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			<td style="width:131px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:111px;">Dissecting scissors</td>
			<td style="width:111px;">World Precision Instruments</td>
			<td style="width:111px;">15922</td>
			<td style="width:131px;"></td>
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			<td height="42" style="height:42px;width:111px;">25 gauge needle</td>
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			<td style="width:111px;">300600</td>
			<td style="width:131px;"></td>
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			<td height="63" style="height:63px;width:111px;">Peristaltic pump</td>
			<td style="width:111px;">Cole-Parmer Instrument Co. Ltd</td>
			<td style="width:111px;">WZ-07522-30</td>
			<td style="width:131px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:111px;">Iris scissors</td>
			<td style="width:111px;">Weiss</td>
			<td style="width:111px;">103227</td>
			<td style="width:131px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:111px;">No.2 tweezers</td>
			<td style="width:111px;">World Precision Instruments</td>
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			<td style="width:131px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:111px;">Coronal Brain Matrix</td>
			<td style="width:111px;">World Precision Instruments</td>
			<td style="width:111px;">RBMS-200C</td>
			<td style="width:131px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:111px;">Razor blade</td>
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			<td style="width:131px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:111px;">OCT medium</td>
			<td style="width:111px;">Tissue tek</td>
			<td style="width:111px;">4583</td>
			<td style="width:131px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:111px;">Cryostat/microtome</td>
			<td style="width:111px;">Bright</td>
			<td style="width:111px;"></td>
			<td style="width:131px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:111px;">Superfrost plus slides</td>
			<td style="width:111px;">Thermo Scientific</td>
			<td style="width:111px;">J1800AMNZ</td>
			<td style="width:131px;"></td>
		</tr>
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			<td height="42" style="height:42px;width:111px;">Sodium citrate</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:111px;">S4641</td>
			<td style="width:131px;">for 65&#176;C wash buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:111px;">Formamide</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:111px;">F7503</td>
			<td style="width:131px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:111px;">Tween-20</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:111px;">P1379</td>
			<td style="width:131px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:111px;">Coverslips</td>
			<td style="width:111px;">VWR International</td>
			<td style="width:111px;">631-0146</td>
			<td style="width:131px;"></td>
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			<td height="42" style="height:42px;width:111px;">Coplin Jar</td>
			<td style="width:111px;">Smith Scientific Ltd</td>
			<td style="width:111px;">2959</td>
			<td style="width:131px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:111px;">Blocking reagent</td>
			<td style="width:111px;">Roche</td>
			<td style="width:111px;">11096176001</td>
			<td style="width:131px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:111px;">Heat-inactivated sheep serum</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:111px;">S2263</td>
			<td style="width:131px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:111px;">Hydrophobic pen</td>
			<td style="width:111px;">Cosmo Bio</td>
			<td style="width:111px;">DAI-PAP-S</td>
			<td style="width:131px;">1:500</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:111px;">&#945;-FITC POD-conjugated antibody</td>
			<td style="width:111px;">Roche</td>
			<td style="width:111px;">11426346910</td>
			<td style="width:131px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:111px;">TSA&#8482; Plus Fluorescein System</td>
			<td style="width:111px;">Perkin Elmer</td>
			<td style="width:111px;">NEL741001KT</td>
			<td style="width:131px;">1:1500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:111px;">&#945;-DIG AP-conjugated</td>
			<td style="width:111px;">Roche</td>
			<td style="width:111px;">11093274910</td>
			<td style="width:131px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:111px;">Fast red tablets</td>
			<td style="width:111px;">Roche</td>
			<td style="width:111px;">11496549001</td>
			<td style="width:131px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:111px;">.22&#181;M filter</td>
			<td style="width:111px;">Millex</td>
			<td style="width:111px;">SLGP033RS</td>
			<td style="width:131px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:111px;">&#945;-Olig2 Rabbit antbody</td>
			<td style="width:111px;">Millipore</td>
			<td style="width:111px;">AB9610</td>
			<td style="width:131px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:111px;">Alexa Fluor&#174; 647-conjugated &#945;-rabbit antibody</td>
			<td style="width:111px;">Life technologies</td>
			<td style="width:111px;">A-31573</td>
			<td style="width:131px;">1:1000</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:111px;">bisBenzimide H 33258</td>
			<td style="width:111px;">sigma</td>
			<td style="width:111px;">B2883</td>
			<td style="width:131px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:111px;">Mounting medium</td>
			<td style="width:111px;">Dako</td>
			<td style="width:111px;">S3023</td>
			<td style="width:131px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:111px;">Leica SP2 confocal microscope</td>
			<td style="width:111px;">Leica</td>
			<td style="width:111px;"></td>
			<td style="width:131px;"></td>
		</tr>
	</tbody>
</table>

combining double fluorescence in situ hybridization with immunolabelling for detection of the expression of three genes in mouse brain sections

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.

This article describes a method for a double fluorescence ISH followed by immunolabelling in mouse brain sections. A brief description of this protocol is shown in Figure 1.&#160;The first step was to synthesize probes specific to Aspa and Mbp (myelin basic protein). To check that the probes had been synthesized, a small aliquot of each reaction was run on an agarose gel. The faint linear template and a large amount of the RNA probe can be seen (...

Watch this Scientific Journal Video about Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections at JoVE.com

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

The overall goal of this experiment is to simultaneously detect the expression of three different genes in brain sections, using double fluorescence in situ hybridization followed by immunostaining. This method can help answer key questions in the neuroscience field, such as which cell type expresses my gene of interest? The main advantage of this technique is that you don't need a good antibody for your gene of interest to be able to localize its expression to a specific cell type.To summarize this procedure, on the first day, fixed tissue sections are cut and hybridized with two differently-labeled RNA probes overnight. On the second day, the sections are incubated overnight with an antibody targeting the FITC labeled probe. This antibody is conjugated with horseradish peroxidase, which catalyzes the addition of a fluorescent Tyramide on the third day.Then, an alkaline phosphatase conjugated antibody is used to target the DIG labeled probe. On day four, this enzyme catalyzes the fluorescent precipitate formation when incubated with Fast Red solution. Then the sections are incubated overnight with a primary antibody targeting a protein of interest.Finally, on day five, this protein is visualized with a fluorophore conjugated secondary antibody. To begin this procedure, choose a DNA clone that contains the cDNA sequence of the gene of interest in a plasmid with RNA polymerase promoters. Next, grow the DNA clone, extract the plasmid with a small scale plasmid purification kit, and sequence it with a T7 and SP6 universal primers.Digest 10 to 20 micrograms of plasmid DNA with a restriction enzyme that cuts at the five prime end of the sense strand of the cDNA. Incubate it for 1.5 hours at 37 degrees Celsius. Then run a small aliquot on an agarose gel to check that the plasmid is fully linearized.To purify the linearized plasmid, add 1/10 of the volume of three molar sodium acetate, one volume of 10 millimolar Tris-HCl equilabrated phenol, and one volume of chloroform isoamyl alcohol. Centrifuge at 16, 000 Gs for one minute at room temperature, and extract the upper aqueous phase. Then add one volume of chloroform IAA.Centrifuge it at 16, 000 Gs for one minute, and extract the upper aqueous phase. Repeat this step once again. Then, add two volumes of ethanol, and keep it at negative 20 degrees Celsius for one hour, or overnight.After that, centrifuge at 16, 000 Gs at four degrees Celsius for 10 minutes. Discard the supernatant, and wash the pellet with 70%cold ethanol. Then re-suspend it in TE at a concentration of one microgram of cDNA per 2.5 microliters.To synthesize the two probes, first select the appropriate RNA polymerase. Then, prepare the in vitro transcription reaction, and bring the final volume up to 20 microliters with DEPC-treated water. Incubate at 37 degrees Celsius for 1.5 hours.Subsequently, run one microliter of the transcription reaction on a 1%agarose gel to check if the reaction has worked. The gel should show the linear template and a bright band of the probe. In this procedure, cut 15 micrometer thick sections of frozen tissue in a cryostat.Collect the sections onto the microscope slides, and leave the slides to dry for one hour to ensure the tissues adhere to the slides. The most important factor for this procedure to work well is the quality of the sections. This depends partly on having well-perfused and fixed tissue, and partly on sectioning technique to obtain flat sections that are free of RNase contamination.Then, dilute the two probes in hybridization buffer pre-warmed to 65 degrees Celsius, and mix well. Apply 300 microliters of hybridization mixture onto each slide, and cover with an oven-baked cover slip. Subsequently, incubate the slides overnight at 65 degrees Celsius in a humidified chamber.The next day, transfer the slides into a Coplin jar containing pre-warmed wash buffer, and wash the slides for 30 minutes, twice, at 65 degrees Celsius. After that, wash the slides for 10 minutes three times with MABT wash solution at room temperature. In this step, use a hydrophobic pen to draw circles around the tissue sections.Then incubate the slides for one hour at room temperature with ISH blocking buffer in a humidified chamber. Next, incubate the slides overnight at 4 degrees Celcius with a horseradish peroxidized conjugated anti-fitc antibody. Then, place the slides into a coplin jar containing PBST.Wash them in PBST three times for 10 minutes each time, and replace with fresh PBST each time. Afterward, add fluorescent tiromide to the slides and leave them for 10 minutes at room temperate. Place the slides into a coplin jar and wash in PBST for 10 minutes three times.Subsequently, incubate the slides with ISH blocking buffer for one hour at room temperature. Then, incubate the slides overnight at four degrees Celcius with an alkaline phosphatase conjugated anti-dig antibody. After that, wash them with MABT three times for 10 minutes each time.Next, wash the slides twice with 0.1 molar tris HCL at pH 8.2 for five minutes at room temperature. Filter the fast red solution, and incubate the slides at 37 degrees Celcius with fast red solution in a humidified chamber. As the time for optimal development varies, check the slides regularly with a fluorescent microscope to monitor the formation of precipitate.Then, wash them three times with PBST for 10 minutes each time. To perform immunohistochemistry, incubate the slides with IHC blocking buffer for one hour at room temperature in a humidified chamber. Next, incubate the slides in the primary antibody solution at four degrees Celsius overnight.The next day, wash the slides with PBST for 10 minutes three times. Incubate them in the secondary antibody solution at room temperature for one hour. Then, wash them with PBST three times for 10 minutes each time.Subsequently, partially dry the slides at room temperature and mount with a fluorescence mounting medium. Leave the slides to dry before imaging them in a confocal microscope. Shown here are the representative images from ISH for Aspa and Mbp, followed by immuno-staining for OLIG2 combined with hook-staining.Aspa was expressed in some MBP-positive OLIG2-positive cells in the cortex and in the corpus callosum. Some Mbp-positive OLIG2-positive cells do not express Aspa. While performing this procedure, it is important to remember to keep the slices and all the solutions free of RNase contamination.After watching this video, you should have a good understanding of how to study gene expression patterns by N-citrine inhibization and immunohistochemistry. This technique can be adapted for a variety of tissue of different origins and developmental stages.

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Research

JoVE Journal

Neuroscience

12.8K vistas.  University College London. The overall goal of this experiment is to simultaneously detect the expression of three different genes in brain sections, using double fluorescence in situ hybridization followed by immunostaining. This method can help answer key questions in the neuroscience field, such as which cell type expresses my gene of interest? The main advantage of this technique is that you don't need a good antibody for your gene of interest to be able to localize its expression to a specific cell type.To ...