Name: detection of the genome and transcripts of a persistent dna virus in neuronal tissues by fluorescent in situ hybridization combined with immunostaining
Uploaded: 2014-01-23T14:45:00
Description: Watch this Scientific Journal Video about Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining at JoV

We thank N. Sawtell (Cincinnati Children&#8217;s Hospital Medical Center, Cincinnati, Ohio, USA), S. Efstathiou (University of Cambridge, UK) and James Hill (LSU Health Sciences Center, New Orleans, USA) for providing samples from HSV-1-infected mice and rabbits, respectively, and for reagents; H. Masumoto (Kazusa DNA Research Institute, Chiba, Japan) and S. Khochbin (Institut Albert Bonniot, Grenoble, France) for helpful discussions.
This work was funded by grants from the Centre National de la Recherche Scientifique (CNRS) (ATIP program, to PL, http://www.cnrs.fr), the French National Research Agency (ANR) (ANR-05-MIIM-008-01, CENTROLAT, <a href="http://www.agencenationale-recherche.fr">http://www.agencenationale-recherche.fr</a>), the FINOVI Foundation (<a href="http://www.finovi.org/:fr:start">http://www.finovi.org/:fr:start</a>), the LabEX DEVweCAN (ANR-10-LABX-61) of Universit&#233; de Lyon, within the program &#34;Investissements d&#39;Avenir&#34; (ANR-11-IDEX-0007) operated by the ANR (<a href="http://www.agence-nationale-recherche.fr">http://www.agence-nationale-recherche.fr</a>), l'Association pour la Recherche contre le Cancer (ARC-7979 and ARC-4910, <a href="http://www.arc-cancer.net">http://www.arc-cancer.net</a>), la Ligue Nationale Contre le Cancer (LNCC, <a href="http://www.ligue-cancer.net">http://www.ligue-cancer.net</a>), and INCa (EPIPRO program, <a href="http://www.e-cancer.fr">http://www.e-cancer.fr</a>). FC and PL are CNRS researchers.

This method was used in a study published previously 22. For general background and description of conventional manipulation on ISH, IF and FISH, we suggest the following available literature 23.
1. Animal Infection
All procedures involving experimental animals conformed to ethical issues from the Association for Research in Vision and Ophthalmology (ARVO) Statement for the use of animals in research, and were approved by the local Ethical Commit...

The protocol described here allows the detection of HSV-1 latent genome within neurons of mouse neuronal tissue sections. Our understanding of the pathways regulating viral gene expression has been limited by the lack of method to detect HSV-1 genomic DNA in situ within neuronal tissues. Information on genome copy number and proportion of infected neurons came mainly from PCR analysis on dissociated neurons11,12. In elucidating the role the host-cell nuclear architecture on HSV-1 latency, we set to de...

Herpes simplex virus type 1 (HSV-1) is a persistent human neurotropic virus, establishing a long-term latent infection in neurons of the trigeminal ganglia (TG) of the peripheral nervous system, from which it reactivates periodically to replicate and spread. The HSV-1 genome is a 150 kb dsDNA localizing in the nucleus of the host neuron where it remains as multicopy chromatinized plasmids, which do not integrate into the host-cell genome1,2. During latency, the HSV-1 replicative cycle genetic program is strongly repressed, and gene expression is restricted to the latency-associated transcript (LAT) locus, from latency establishment to initiation of reactivation3. LAT produces a long 8.5 kb noncoding RNA processed into a major 2 kb stable lariat, and several miRNA4-7. HSV-1 latency is thus characterized by the presence of the viral genomic DNA, LAT RNA, and the absence of detectable replicative cycle proteins.
Animal models, predominantly mouse and rabbit, are experimental models recapitulating several features of latency in human. One of the main interests of those models is that they allow studying physiological aspects of HSV-1 latency in immunocompetent hosts. Over the past decades, many experimental tools, such as genetically modified viruses and mice, have been developed to study the physiology, genetics, and cellular biology of HSV-1 latency, from animal tissues. Until now, viral genomic DNA was detected and quantified by Southern blot and qPCR from dissociated TGs. However, there is currently no method available to detect HSV-1 genome by in situ hybridization on tissue sections8. Consequently, latency is routinely assessed on histological sections through the detection of LAT RNA by RNA in situ hybridization rather than viral genome detection. Because it has been impossible to characterize infected cells based on the presence of viral genomes, this technical limitation has been a major drawback to the analysis of many aspects of the host-virus interactions, such as the relationship between the viral genome and cellular and viral gene expression&#160;or the host cell-mediated immune response9,10.
Most importantly, the cell-to-cell heterogeneity of the latent infection remains relatively unexplored and has been shown to be a key feature of latency in mice and in human sensory ganglion neurons implanted into SCID mice11-17. Typically, it was shown by qPCR that the HSV-1 genome copy number per cell varies from 5 to several hundreds. Although LAT appears as a key regulator of latency and reactivation, qPCR data on isolated neurons and in situ PCR indicated that only a subset of latently infected neurons, as low as 30%, expresses the LAT locus11,12,18-21. How the host cell and the cellular environment within the tissue impact on the virus latency establishment and viral gene expression remains unclear. Here we describe a robust fluorescent in situ hybridization (FISH) method for the efficient detection of low-copy HSV-1 genomic DNA within animal neuronal tissue sections. This method has been designed and used by us to get access to high resolution microscopy imaging that is necessary to study the interaction of the viral genome with the host cell intra-nuclear components22. Additionally, we describe a multiple staining method for the simultaneous detection of the viral DNA with RNA and proteins, which is a unique tool to describe the virus-host interactions that regulate viral gene expression. The method can also be applied for a broad range of analyses requiring the detection of HSV-1 latent genome, such as quantifying infected neurons in large number of sections. A key step is to apply antigen retrieval treatment to make the viral DNA accessible to hybridization. Thus, this protocol might also be efficient to the detection of other dsDNA viruses, which are currently not detectable by conventional DNA-FISH approaches within animal tissues.

<table border="0" cellpadding="0" cellspacing="0" width="822">
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	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:203px;">Balb/c mice</td>
			<td style="width:189px;">Janvier, France</td>
			<td style="width:93px;"> </td>
			<td style="width:337px;">6 week-old females</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;">HSV-1 strains</td>
			<td>SC16 strain (wild type)</td>
			<td></td>
			<td style="width:337px;">See Labetoule, M. et al. (2003) Invest Ophthalmol Vis Sci 44: 217&#8211;225, for details on HSV-1 strain and virus stock preparation.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ketamine hydrochloride</td>
			<td>Sigma</td>
			<td>K2753</td>
			<td rowspan="2" style="width:337px;">Intraperitoneal injection of a solution containing Ketamine (100mg/kg) and Xylazine (10mg/kg)</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Xylazine hydrochloride</td>
			<td>Sigma</td>
			<td>X1251</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;">Paraformaldehyde (PFA)</td>
			<td>Sigma</td>
			<td>158127</td>
			<td style="width:337px;">Suspend 4g of PFA in 90mL of water. Add 50&#181;L of 1N NaOH, and heat at 60&#176;C in a water bath with agitation. PFA dissolves in about 30min. Add 10mL of 10X PBS. This solution can be prepared in advance and stored at -20 &#176;C in 5mL tubes. Caution. Manipulate under a fume hood.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Physiological Saline</td>
			<td>Sigma</td>
			<td colspan="2">07982-100TAB-F</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">1X PBS, pH 7.4 (sterile)</td>
			<td>Life Technologies</td>
			<td>10010-015</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sucrose</td>
			<td>Sigma</td>
			<td>84100</td>
			<td>Prepare a 20% sucrose solution in 1X PBS.</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:203px;">Cryosectionning embedding medium - Tissue-Tek OCT Compound -</td>
			<td>SAKURA</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Large vector DNA purification kit</td>
			<td>Qiagen</td>
			<td>12462</td>
			<td style="width:337px;">To purify Cosmid or BAC vector containing HSV-1 genome and store at -20&#176;C</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Nick translation kit</td>
			<td>Roche Applied Sciences</td>
			<td>10 976 776 001</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cy3-dCTP</td>
			<td>GE Healthcare</td>
			<td>PA53021</td>
			<td>Protect from light</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">0.5M EDTA</td>
			<td>Sigma</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">G50 Mini spin column</td>
			<td>GE Healthcare</td>
			<td>27-5330-01 </td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Salmon sperm DNA 10mg/mL</td>
			<td>Invitrogen / Life Technologies</td>
			<td>15632-011</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ethanol molecular biology grade</td>
			<td>Sigma</td>
			<td>87047</td>
			<td>Prepare a 70% solution</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Salmon sperm DNA</td>
			<td>Invitrogen / Life Technologies</td>
			<td>15632-011</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Formamid Molecular biology grade</td>
			<td>Sigma</td>
			<td>F9037</td>
			<td>Caution. Manipulate under fume hood.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">HSV-1 biotinylated commercial probe </td>
			<td>Enzo Life Sciences</td>
			<td>ENZ-40838</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">ImmEdge hydrophobic pen</td>
			<td>Vector Laboratories</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">20X Saline Sodium Citrate (SSC)</td>
			<td>Sigma</td>
			<td>S6639</td>
			<td>Prepare a 2X SSC solution in ddH20.</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td style="width:337px;">Prepare a 10% stock solution in water and store at +4&#176;C. Prepare the 0.5% solution in 1X PBS right before use.</td>
		</tr>
		<tr height="102">
			<td height="102" style="height:102px;">10mM sodium citrate pH 6.0</td>
			<td>Sigma</td>
			<td>S1804</td>
			<td style="width:337px;">Prepare a 100mM stock solution (10X). Weigh 10,5g of citric acid (MW 210.14. Caution, irritant and toxic, wear appropriate mask and gloves), and dissolve in 400mL water. Adjust pH at 6.0 with 1N NaOH (caution, irritant, wear gloves). Adjust to 500mL with distilled water. Dilute 10 times in distilled water before use.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Acetic Acid</td>
			<td>Sigma</td>
			<td>320099</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Methanol, molecular biology grade</td>
			<td>Sigma</td>
			<td>322415</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dextran sulfate - MW 500 000</td>
			<td>Euromedex</td>
			<td>EU0606-A</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Denhardt&#39;s solution (100X)</td>
			<td>Euromedex</td>
			<td>1020-A</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Rubber Cement &#34;FixoGum&#34;</td>
			<td>Marabut</td>
			<td>290110000</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:203px;">DNA purification kit - Qiaquick PCR purification kit -</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">T7 in vitro transcription kit</td>
			<td>Ambion / Life Technologies</td>
			<td>AM1314</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Biotin-16-UTP</td>
			<td>Roche Applied Sciences</td>
			<td>11388908910</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">RNA purification mini-column</td>
			<td>Qiagen</td>
			<td>73404</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ribonucleoside Vanadyl Complex</td>
			<td>New England Biolabs</td>
			<td>S1402S</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">H2O2</td>
			<td>Sigma</td>
			<td>H3410</td>
			<td style="width:337px;">Prepare a 3% solution in distilled water. Store at +4 &#176;C and protect from light.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Yeast tRNA</td>
			<td>Invitrogen</td>
			<td>15401011</td>
			<td style="width:337px;">prepare a 10mg/mL solution in RNAse free water</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Normal Goat Serum</td>
			<td>Invitrogen</td>
			<td>PCN5000</td>
			<td></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;">Primary antibodies</td>
			<td>Any supplier</td>
			<td></td>
			<td style="width:337px;">The following primary antibodies were used in the result section: anti-mouse CENP-A (rabbit mAb C51A7, Cell Signaling Technologies), and anti-ATRX H-300 (Santa Cruz Biotechnology)</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;">Secondary fluorescent antibodies</td>
			<td>Invitrogen / Life Technologies</td>
			<td></td>
			<td style="width:337px;">The fluorescent secondary antibodies routinely used in our protocol are AlexaFluor labeled goat antibodies (IgG H+L). The antibody used in the result section is an anti-rabbit goat antibody labaled with AlexaFluor 488 (reference A11001)</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:203px;">Tyramide Signal Amplification (TSA) kit - Streptavidin + AlexaFluor 350 (blue fluorescence)</td>
			<td>Invitrogen / Life Technologies</td>
			<td>#T20937</td>
			<td rowspan="2" style="width:337px;">TSA kits are also available from Perkin Elmer</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:203px;">Tyramide Signal Amplification (TSA) kit - Streptavidin + AlexaFluor 488 (green fluorescence)</td>
			<td>Invitrogen / Life Technologies</td>
			<td>#T20932</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Hoechst 33342</td>
			<td>Invitrogen / Life Technologies</td>
			<td>H3570</td>
			<td style="width:337px;">Prepare a 0.5&#181;g/mL solution in 1X PBS immediatly before use. Discard the remaining solution.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">22x50mm coverslip. n&#176;1.5 glass.</td>
			<td>Electron Microscopy Sciences</td>
			<td>72204-04</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:203px;"> Mounting medium with anti-fading agent - Vectashield -</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td style="width:337px;">Another conventional product is Fluoromount G from electron microscopy Science</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Superfrost glass slides</td>
			<td>FisherScientific</td>
			<td>12-550-15</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;"> </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">EQUIPMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">Equipment / material</td>
			<td>Company</td>
			<td>Reference</td>
			<td>Note</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Needle for infection</td>
			<td></td>
			<td></td>
			<td>Glass micropipette hot drawn. Home made.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dissection equipement</td>
			<td>Moria, France</td>
			<td></td>
			<td>Microsurgical scissors and forceps</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Peristaltic pump</td>
			<td style="width:189px;">Cole Palmer Instruments</td>
			<td></td>
			<td>Easyload Masterflex</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Micro-syringe pump device (Nano Pump)</td>
			<td>kdScientific</td>
			<td>KDS310</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cryostat</td>
			<td>Leica France</td>
			<td>CM 1510-1</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">-80 &#176;C freezer</td>
			<td>Sanyo</td>
			<td></td>
			<td>Ultra Low -80&#176;C</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Domestic microwave oven</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dry block heater</td>
			<td>Eppendorf</td>
			<td>022670204</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Incubator Slide moat</td>
			<td>Boekel Scientific</td>
			<td>240000</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Coplin Jar</td>
			<td>Dominique Dutscher</td>
			<td>68512</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Staining glass container</td>
			<td>Dominique Dutscher</td>
			<td>68506</td>
			<td></td>
		</tr>
		<tr height="136">
			<td height="136" style="height:136px;">Fluorescent microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td style="width:337px;">The images presented in the result section were collected with a Zeiss AxioObserver with objective x40 LD NeoFluor N.A 0.6, and x100 PlanApochromat N.A 1.3. Filter set #38, #43 and #43. HXP 120 fluorescence light source. Photometrics CoolSNAP HQ2 CCD camera. Signal will be more easily observed on a recent high efficiency microscope such as Zeiss AxioImager/AxioObserver series, Nikon Ti-E/Ni-E series or Leica DM/DMI6000 series</td>
		</tr>
	</tbody>
</table>

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.

After several months of extensive testing, we discovered that heat-based chemical unmasking made latent HSV-1 genome available for fluorescent in situ hybridization. During the process, we tried various unmasking procedures, and only heat-based treatments (i.e. heating the sections up to sub-boiling temperature in a microwave oven) appeared efficient. We then tested several salt buffers that are routinely used in immunohistochemistry (IHC) and electron microscopy to retrieve epitopes31,32, in...

Watch this Scientific Journal Video about Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining at JoV

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.

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 ...

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Double Fluorescence in situ Hybridization in Fresh Brain Sections

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Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

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

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 ...

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 ...