Name: combining double fluorescence in situ hybridization with immunolabelling for detection of the expression of three genes in mouse brain sections
Uploaded: 2016-03-26T15:00:00
Description: 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

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>
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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.

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			<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.

Research

JoVE Journal

Neuroscience

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal.  Such analysis of ...

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within ...

Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization

Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization

This study describes a protocol for the multiplex in situ hybridization (ISH) of the mouse jugular-nodose ganglia, with a particular emphasis on detecting ...

Deep-Tissue Three-Photon Fluorescence Microscopy in Intact Mouse and Zebrafish Brain

Multiphoton microscopy techniques, such as two-photon microscopy (2PM) and three-photon microscopy (3PM), are powerful tools for deep-tissue in vivo ...