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

The overall goal of this procedure is to detect the genome of herpes simplex virus, type one in sections of trigeminal ganglia from latently infected mice by fluorescent in situ hybridization. This is accomplished by first inoculating the virus in the animal lip, followed by a 28 day incubation period during which the virus gets established in the trigeminal ganglia. Next, the trigeminal ganglia are harvested, embedded, and cryo sectioned.

The sections are subjected to several preparatory treatments, including the key step of heating them in a sodium citrate buffer. Finally, fluorescent in situ hybridization is performed using herpes specific fluorescent robes. Ultimately, results can show the positioning of the viral genome inside the nucleus of individually infected neurons through the use of confocal microscopy.

In many cases, the study of a various lifecycle requires the use of animal models. The main benefit of the technique represent here is that it provides data at the single cell level using an institute fluent ization approach. The royal killer model of HS infections reproduces most of the aspects of the natural history of HSV one infection in humans.

That is the natural host of the virus. The primary infection is made in the oral tissues, and then the virus progresses from the lips to the two al ganglion. That is the main side of HS V latency in humans Combined two immuno and staining.

This method can help answer key questions concerning herpes virus latency, such as how the virus interacts with nuclear components, and how these interactions would influence the maintenance of latency and eventually reactivation of the virus. To begin, have an anesthetized mouse and an HSV one virus stock solution. Resuspended in phenol red free medium.

Using a binocular stereoscope, insert the needle in the subepithelial layer of the left upper lip at the mucocutaneous border. Inject 0.5 microliters of virus solution over five seconds, and then make a second injection of virus at the same site. Transfer the mouse to a 37 degree Celsius incubator or heating pad until it awakens.

Then put it in its home cage for the required recovery time before perfu fixing it according to the text protocol. Cut on each side of the lower jaw. Then cut the tip of the nose just behind the incisors to reveal the nose cavity.

Cut the palate in half with scissors, and then place it to the side. The trigeminal ganglia are beneath the palate. They're white oblong masses, two to three millimeters long and are connected to the trigeminal nerve.

Cut the trigeminal nerve branches on each side of the ganglia to release them from the brain stem. Using surgical pliers, remove the ganglia and transfer them to 20%Sucrose in PBS allow them to incubate for 24 hours. At room temperature the next day, embed both trigeminal ganglia in a single block of cryosectioning.

Embedding medium, freeze the block at minus 80 degrees Celsius until it is sectioned on a cryostat. Cut the trigeminal ganglia lengthwise into 10 micron sections and collect them on super frost Slides warmed to 30 degrees Celsius. Four to five sections can fit on a single slide.

Giving the slide multiple series labels can be useful. If several downstream applications are planned, let the slices dry for five to 10 minutes before storing them at minus 80 degrees Celsius. For this protocol, the probe is prepared by labeling cosms containing 30 kilobase portions of the HSV one genome with SI 3D CTP by NIC translation.

It is precipitated and suspended in deionized form amide and should appear pink due to S SI three incorporation on day one. Place the slides on a slide holder at room temperature and let the sections dry for 10 minutes. Then rehydrate the sections in one XPBS for 10 minutes.

Next, perme the tissue in 0.5%Triton X 100 in one XPBS for 20 minutes. Then wash the slides three times for 10 minutes per wash with two XSS and keep them in two XSSC until the unmasking buffer is heated. For unmasking preheat the citrate buffer in a microwave until the buffer boils.

This is done by filling a glass slide tray with 200 milliliters of the buffer. Place the tray in a larger container filled with 500 milliliters of distilled water and preheat the buffer until boiling. Then transfer the slides to the tray.

Verify that they're completely covered with buffer in the microwave. Heat the slides in the buffer for about 20 seconds until the buffer reaches boiling. Do not let the buffer boil over.

Then cool down the slides at room temperature for two minutes and repeat the heating cycle six more times. It is critical to empirically determine the number and duration of eating cycles for the reproducibility of the unmasking step. The microwave oven, the tray, the container, the volume of buffer in the tray.

The volume of water in the container should be kept identical When the heating is finished. Transfer the slides in two XSSC for five minutes under a fume hood, incubate the slides in a three to one to four solution of methanol, acetic acid and PBS for 15 minutes. Then incubate the slides in a freshly prepared three to one methanol to acetic acid mix for 15 minutes.

Now dehydrate the sections through successive 10 minute incubations in 70%ethanol, followed by two 10 minute incubations in pure ethanol. Let the slides dry at room temperature for 10 minutes to prepare them for the probe. Apply 80 microliters of probing solution to the dried sections dropwise and cover them with a cover slip.

Check that the probing solution spreads over the entire surface of the cover slip and that there are no bubbles. Then seal the cover slip with rubber cement and let it dry. Proceed with denaturation by incubating the slides at 80 degrees Celsius for five minutes.

Then quickly transfer the slides onto a metallic tray on ice for five minutes. Follow this with an overnight hybridization at 37 degrees Celsius the next day. Remove the rubber cement with forceps with the slides on a 37 degree Celsius heat block.

Remove the cover slip gently with the tip of a scalpel blade. Next, wash the sections repeatedly with SSC. Now, stain the samples for 10 minutes with the appropriate dye like DAPI or hooks 3 3 3 4 2 at 0.5 micrograms per milliliter in one XPBS.

Then wash the slides three times with one XPBS for 10 minutes per wash. After the third wash, drain as much liquid as possible from the slide. Then at the end of each slide, apply 80 microliters of mounting medium with an anti fading agent.

Slowly cover the medium with a high optical quality cover slip, avoiding bubble formation. Then seal the slide with nail polish and store it in the dark at four degrees Celsius. In developing this protocol, several salt buffers were tested for unmasking.

While EDTA buffers tended to damage the tissue. Sodium citrate tris, HCL and distilled water were suitable to HSV one detection to verify that the protocol would work with commercial probes. HSV one latent genome detection was performed using a PAN HSV one biotinylated probe obtained from Enzo Biochem, both single and multiple spot patterns for HSV one genome could be detected.

Furthermore, the DNA fish protocol was tested on mice and rabbit infected with three commonly used HSV one strains. In all cases, the HSV one genomes showed a spotty signal with variable brightness and intensity. In addition, the applicability of the protocol was tested in the replicative cycle of HSV one by performing DNA fish on tissues from mice undergoing a general herpes infection.

In these animals, large and bright aggregates of HSV one genomes were detected in various tissues, including the DRG eyes and brain. The DNA fish protocol is very versatile. It allows for co detection of HSV one genome and viral or cellular RNAs and proteins.

For example, co detection of HSV one genome together with the HSV one lat RNA is feasible co detection of HSV one genome with cellular proteins such as the Centro MERIC protein CE NPA A was utilized co detection of HSV one with the chromatin and PML nuclear bodies. Associated protein A TRX were visualized with a triple stain showing HSV 1D NA in red. Its RNA product lat in blue and a TRX in green.

Once unmasking is set up, the DNA fish procedure is very robust. It can be done in less than 24 hours in batches of 20 slides or more. The development of this Technique will enable researchers investigating herpes viruses and other persistent viruses to explore at the single cell level, how cellular components and nuclear architecture could influence the biology of these viruses.