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.

Etikk Uttalelse: Mus drifta og håndtering er i samsvar med britisk hjemmekontoret forskrifter og UCL etiske komité retningslinjer, i samsvar med de Dyr (vitenskapelige prosedyrer) Act 1986 av Storbritannia og dets endringsforskriften 2012. MERK: Alle løsninger bør gjøres med dietylpyrokarbonat (DEPC) - behandlet vann for å ødelegge eventuelle gjenværende RNase. For DEPC behandling, legg DEPC (1 ml per liter), rist kraftig til alle DEPC kulene har forsvunnet da autoklaver å de...

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Denne protokollen gir en trinn-for-trinn fremgangsmåte for en dobbel RNA in situ-hybridisering, etterfulgt av immunfarging. Vi har brukt denne protokollen til å bekrefte at Aspa er uttrykt i modne oligodendrocytter i flere hjerneområder. Denne multi-trinns prosedyre har mange potensielle fallgruver som kan påvirke følsomhet og bør unngås. Først må alle løsninger og lagringsbuffere for transkripsjonsreaksjon må være RNase-fri. For det andre er valget av cDNA temp...

Gliaceller, som er de mest tallrike cellene i sentralnervesystemet (CNS), omfatter oligodendrocytter (de myelinerende celler av CNS), oligodendrocytter forløpere (OPS, også kjent som &quot;NG2 celler&quot;), astrocytter og microglia. Det er økende interesse for funksjonene til gliacellene og deres potensielle roller i nevrologiske sykdommer 1. For eksempel Canavan sykdom (CD) er en arvelig nevrodegenerativ lidelse starter tidlig barndom med spongiform leukodystrophy og et progressivt tap av nerveceller, som fører til døden vanligvis før 10 års alder 2,3. Mutasjoner i Aspartoacyclase (ASPA) genet som fører til drastisk redusert aktivitet ASPA 4 i CD har blitt identifisert. ASPA er et enzymet som katalyserer deacetyleringen av N-acetylaspartate (NAA), et molekyl som sterkt konsentrert i hjernen, genererer acetat og aspartat 5-7. Mange CD-pasienter viser høyere nivåer av NAA grunn av mangel på Aspa activitet. Noen studier spekulerer i at NAA-avledet acetat kan være en viktig kilde av fettsyrer / lipider i hjernen under utvikling og CD kan være resultatet av redusert myelin syntese under utvikling som skyldes svikt i NAA å bli brutt ned 3,5,6. ASPA hovedsakelig funnet i nyre, lever og hvit substans i hjernen, og gitt den viktige rollen ASPA i CD, har den cellulære ekspresjon av dette enzym i hjernen blitt studert av flere laboratorier. Ved å se på Aspa enzymatisk aktivitet i hjernen, tidligere studier har funnet at økningen i Aspa aktivitet under hjernens utvikling paralleller tidsforløpet av myelination 8-10. På cellenivå, analyser analyser for enzymatisk aktivitet, så vel som in situ hybridisering (ISH) og immunhistokjemi (IHC) antyder at ASPA er hovedsakelig uttrykt i oligodendrocytter i hjernen, men ikke i neuroner eller astrocytter 11-16. Noen studier har funnet at Aspa kanskje ogsåuttrykkes i mikroglia i sentralnervesystemet 12,14. Så langt data på Aspa uttrykk i OPS er begrenset. Ifølge en fersk studie der transcriptomes av ulike celletyper i musen hjernebarken inkludert nevroner, astrocytter, ops, nydannede oligodendrocytes, myelinerende oligodendrocytter, microglia, endotelceller, og pericytes ble analysert ved RNA sekvense 17, Aspa utelukkende uttrykt i oligodendrocytes , spesielt i myelinerende oligodendrocytter (http://web.stanford.edu/group/barres_lab/brain_rnaseq.html). Til tross for disse studiene på Aspa uttrykk mønster i hjernen, en rekke usikkerheter forbli. Forskjellige teknikker kan brukes til å studere genuttrykksmønster. IHC er en vanlig brukt metode for å påvise den funksjonelle produkt (dvs. protein) av en genekspresjon i vevssnitt. Til tross for sin flotte verktøyet, har denne teknikken begrensninger som sin søknad og spesifisitet er under to tilgjengeligheten og spesifisiteten av antistoffet er nødvendig. Til sammenligning har ISH fordelen av å være i stand til å avsløre ekspresjon av hvilket som helst gen på mRNA-nivå. Men det kan være teknisk vanskelig å benytte flere prober samtidig for å lokalisere en genekspresjon til spesifikke celletyper. I denne artikkelen beskriver vi en protokoll som kombinerer dobbel fluorescens RNA in situ hybridisering med fluorescens immunomerking av et protein. Vi har brukt dette sett av teknikker for å undersøke ekspresjon mønster av Aspa i musehjerne. Denne metoden gir den nøyaktige studier av genekspresjon ved hjelp av konfokal mikroskopi.

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			<td height="42" style="height:42px;width:111px;">Sodium citrate</td>
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			<td style="width:131px;"></td>
		</tr>
		<tr height="42">
			<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

Påvisning av genuttrykk i forskjellige typer hjerneceller for eksempel, nevroner, astrocytter oligodendrocytes, oligodendrocyte forløpere og microglia, kan bli hemmet av mangel på spesifikke primære eller sekundære antistoffer for immunfarging. Her beskriver vi en protokoll for å påvise ekspresjon av tre forskjellige gener i den samme hjernen delen ved å bruke dobbel fluorescens in situ hybridisering med to genspesifikke prober, etterfulgt av farging med et antistoff med høy spesifisitet rettet mot proteinet kodet for av en tredje genet. Den Aspartoacyclase (ASPA) genet, mutasjoner av noe som kan føre til en sjelden human hvit substans sykdom - Canavan sykdom - antas å bli uttrykt i oligodendrocytter og mikroglia, men ikke i astrocytter og nevroner. Har imidlertid ennå ikke etablert nøyaktig uttrykk mønster av ASPA i hjernen. Denne protokollen har tillatt oss å fastslå at Aspa er uttrykt i en undergruppe av moden oligodendrocytes ennd det kan generelt anvendes på et bredt spekter av genuttrykksmønstrene studier.

Denne artikkelen beskriver en fremgangsmåte for en dobbel fluorescens ISH etterfulgt av immunomerking i musehjerneseksjoner. En kort beskrivelse av denne protokollen er vist i figur 1. Det første trinn var å syntetisere prober som er spesifikke for Aspa og MBP (myelin basisk protein). For å kontrollere at probene var blitt syntetisert, ble en liten alikvot fra hver reaksjon kjørt på en agarosegel. Svak lineære templat og en stor mengde RNA-probe ...

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.

Kombinere Double Fluorescence<em&gt; In Situ</em&gt; Hybridisering med immunomerking for Påvisning av Expression of Three Gener i musehjerne §§

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

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

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

Påvisning av Genome og transkripsjoner av en vedvarende DNA virus i nerve vev ved Fluorescent In situ Hybridisering Kombinert med 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

Påvisning av Axonally Lokalisert mRNAs i hjernedelene med høy oppløsning<em&gt; In Situ</em&gt; Hybridisering

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

Rapid<em&gt; In Situ</em&gt; Hybridisering bruker oligonukleotidprober på Paraformaldehyde-prefiks Brain of Rats med serotonergt syndrom

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

Lokalisering av luktningsmiddelreceptorgener i Locust Antenna av RNA<em&gt; I situ</em&gt; Hybridisering

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

Store tredimensjonale Imaging mobilnettet organisasjon i musen 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

Kombinere multiplex fluorescens in situ hybridisering med fluorescerende immunhistokjemi på ferske frosne eller faste musehjerneseksjoner

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

Påvisning av G Protein-koblet reseptoruttrykk i mus Vagal Afferent Neurons ved hjelp av 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

Dypvev tre-foton fluorescens mikroskopi i intakt mus og sebrafisk hjerne

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