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>
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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.

combining-double-fluorescence-situ-hybridization-with-immunolabelling

Research

JoVE Journal

Neuroscience

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 frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

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

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal. Such analysis of two or more genes needs to be complemented with in situ hybridization of other genes, or immunohistochemistry of their proteins, both in whole mounted developing organs or sections for detailed resolution of the cellular and tissue expression alterations. Combining multiple gene alterations requires the use of cre or flipase to conditionally delete genes and avoid embryonic lethality. Required breeding schemes dramatically enhance effort and cost proportional to the number of genes mutated, with an outcome of very few animals with the full repertoire of genetic modifications desired. Amortizing the vast amount of effort and time to obtain these few precious specimens that are carrying multiple mutations necessitates tissue optimization. Moreover, investigating a single animal with multiple techniques makes it easier to correlate gene deletion defects with expression profiles. We have developed a technique to obtain a more thorough analysis of a given animal; with the ability to analyze several different histologically recognizable structures as well as gene and protein expression all from the same specimen in both whole mounted organs and sections. Although mice have been utilized to demonstrate the effectiveness of this technique it can be applied to a wide array of animals. To do this we combine lipophilic dye tracing, whole mount in situ hybridization, immunohistochemistry, and histology to extract the maximal possible amount of data.

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

A combination of different techniques to maximize data collection from mouse tissue is presented.

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

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

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

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

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

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

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

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

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

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment. Recently, the development of short oligonucleotide probes has simplified in situ hybridization procedures and allowed the use of paraformaldehyde-prefixed brain sections, which are more widely available in laboratories. Here, we describe a detailed protocol using non-radioactive oligonucleotide probes on the prefixed brain tissues. Hybridization probes used for this study include dapB (a bacterial gene coding for dihydrodipicolinate reductase), ppiB (a housekeeping gene coding for peptidylprolyl isomerase B), and htr2a (a serotonin gene coding for 5-HT2A receptors). This method is relatively simply, cheap, reproducible and requires less than two days to complete.

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

This protocol describes a rapid and simplified in situ hybridization method ideal forparaformaldehyde-prefixed brain, thus reducing the need for prolonged complex steps while using fresh frozen tissues. The method is validated using the identification of the serotonin 5-HT2A receptor gene htr2a in rats.

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

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs&#39; membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

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

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

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 properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the 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

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within cells. Neurochemical phenotyping of functionally identified neurons usually requires concurrent labelling with multiple antibodies (targeting protein) using immunohistochemistry (IHC) and optimization of in situ hybridization (targeting RNA), in tandem. A &#34;neurochemical signature&#34; to characterize particular neurons may be achieved however complicating factors include the need to verify FISH and IHC targets before combining the methods, and the limited number of RNAs and proteins that may be targeted simultaneously within the same tissue section.
Here we describe a protocol, using both fresh frozen and fixed mouse brain preparations, which detects multiple mRNAs and proteins in the same brain section using RNAscope FISH followed by fluorescence immunostaining, respectively. We use the combined method to describe the expression pattern of low abundance mRNAs (e.g., galanin receptor 1) and high abundance mRNAs (e.g., glycine transporter 2), in immunohistochemically identified brainstem nuclei.
Key considerations for protein labelling downstream of the FISH assay extend beyond tissue preparation and optimization of FISH probe labelling. For example, we found that antibody binding and labelling specificity can be detrimentally affected by the protease step within the FISH probe assay. Proteases catalyze hydrolytic cleavage of peptide bonds, facilitating FISH probe entry into cells, however they may also digest the protein targeted by the subsequent IHC assay, producing off target binding. The subcellular location of the targeted protein is another factor contributing to IHC success following FISH probe assay. We observed IHC specificity to be retained when the targeted protein is membrane bound, whereas IHC targeting cytoplasmic protein required extensive troubleshooting. Finally, we found handling of slide-mounted fixed frozen tissue more challenging than fresh frozen tissue, however IHC quality was overall better with fixed frozen tissue, when combined with RNAscope.

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

This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

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

This study describes a protocol for the multiplex in situ hybridization (ISH) of the mouse jugular-nodose ganglia, with a particular emphasis on detecting the expression of G protein-coupled receptors (GPCRs). Formalin-fixed jugular-nodose ganglia were processed with the RNAscope technology to simultaneously detect the expression of two representative GPCRs (cholecystokinin and ghrelin receptors) in combination with one marker gene of either nodose (paired-like homeobox 2b, Phox2b) or jugular afferent neurons (PR domain zinc finger protein 12, Prdm12). Labeled ganglia were imaged using confocal microscopy to determine the distribution and expression patterns of the aforementioned transcripts. Briefly, Phox2b afferent neurons were found to abundantly express the cholecystokinin receptor (Cck1r) but not the ghrelin receptor (Ghsr). A small subset of Prdm12 afferent neurons was also found to express Ghsr and/or Cck1r. Potential technical caveats in the design, processing, and interpretation of multiplex ISH are discussed. The approach described in this article may help scientists in generating accurate maps of the transcriptional profiles of vagal afferent neurons.

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

Multiplex in situ hybridization (ISH) was employed to simultaneously visualize the transcripts for two G protein-coupled receptors and one transcription factor in the entire vagal ganglionic complex of the adult mouse. This protocol could be used to generate accurate maps of the transcriptional profiles of vagal afferent neurons.

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 imaging with subcellular resolution. 3PM has two major advantages for deep-tissue imaging over 2PM that has been widely used in biology laboratories: (i) longer attenuation length in scattering tissues by employing ~1,300 nm or ~1,700 nm excitation laser; (ii) less background fluorescence generation due to higher-order nonlinear excitation. As a result, 3PM allows high-contrast structural and functional imaging deep within scattering tissues such as intact mouse brain from the cortical layers to the hippocampus and the entire forebrain of adult zebrafish.
Today, laser sources suitable for 3PM are commercially available, enabling the conversion of an existing two-photon (2P) imaging system to a three-photon (3P) system. Additionally, multiple commercial 3P microscopes are available, which makes this technique readily available to biology research laboratories. This paper shows the optimization of a typical 3PM setup, particularly targeting biology groups that already have a 2P setup, and demonstrates intravital 3D imaging in intact mouse and adult zebrafish brains. This protocol covers the full experimental procedure of 3P imaging, including microscope alignment, prechirping of ~1,300 and ~1,700 nm laser pulses, animal preparation, and intravital 3P fluorescence imaging deep in adult zebrafish and mouse brains.

Three-photon microscopy enables high-contrast fluorescence imaging deep in living biological tissues, such as mouse and zebrafish brains, with high spatiotemporal resolution.

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